Sample records for comparative lc-ms proteomics

  1. Integration of two-dimensional LC-MS with multivariate statistics for comparative analysis of proteomic samples

    NARCIS (Netherlands)

    Gaspari, M.; Verhoeckx, K.C.M.; Verheij, E.R.; Greef, J. van der


    LC-MS-based proteomics requires methods with high peak capacity and a high degree of automation, integrated with data-handling tools able to cope with the massive data produced and able to quantitatively compare them. This paper describes an off-line two-dimensional (2D) LC-MS method and its

  2. Comparative proteome analysis of wild-type and klotho-knockout mouse kidneys using a combination of MALDI-IMS and LC-MS/MS. (United States)

    Fujino, Yoko; Minamizaki, Tomoko; Hayashi, Ikue; Kawakami, Asako; Miyaji, Takaaki; Sakurai, Kaoru; Yoshioka, Hirotaka; Kozai, Katsuyuki; Okada, Mitsugi; Yoshiko, Yuji


    Mutation of the klotho gene in mice elicits a syndrome resembling accelerated human aging. However, there is limited evidence for the role of Klotho in the kidney. We conducted a comparative proteome analysis of wild-type (WT) and klotho-knockout (kl -/- ) mouse kidneys to identify proteins involved in Klotho deficiency. MALDI imaging MS (MALDI-IMS) of frozen kidney sections from 7-wk-old male WT and kl -/- mice was used to determine genotype-specific differences in the MS distribution. Proteins uniquely distributed in kl -/- kidneys were identified by subsequent analysis of adjacent trypsinized sections by MALDI-IMS in combination with LC-MS/MS. Immunohistochemistry and western blotting were adopted in qualitative and quantitation analysis. Ninety-seven and 69 proteins identified by LC-MS/MS were matched to the MALDI-IMS spectra in WT and kl -/- mouse kidneys, respectively. Among protein types matched, nucleic acid binding proteins were most abundant, followed by enzymes. We identified secretogranin-1 (SCG1), which was predominately distributed in the glomeruli and renal tubules of kl -/- mouse kidneys. Immunohistochemistry for SCG1 mirrored images of MALDI-IMS. SCG1 may be a candidate protein involved in Klotho deficiency. Although further research is needed to investigate the role of SCG1 in the kidney, we show the usefulness of MALDI-IMS combined with LC-MS/MS. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Comparative proteomic analysis of latex from Hevea brasiliensis treated with Ethrel and methyl jasmonate using iTRAQ-coupled two-dimensional LC-MS/MS. (United States)

    Dai, Longjun; Kang, Guijuan; Nie, Zhiyi; Li, Yu; Zeng, Rizhong


    Ethrel (ET) is an effective and widely used latex yield stimulant of Hevea brasiliensis (Pará rubber tree), and jasmonate (JA) is a key inducer of laticifer differentiation in this plant. To examine variations in the latex proteome caused by these phytohormones, ET and methyl jasmonate (MeJA) were applied to Reyan 7-33-97 rubber tree clones, and comparative proteomic analyses were conducted. On the basis of a transcriptome shotgun assembly (TSA) sequence database and an iTRAQ-coupled two-dimensional LC-MS/MS approach, 1499 latex proteins belonging to 1078 clusters were identified. With a 1.5-fold cut-off value to determine up- and down-regulated proteins, a total of 101 latex proteins were determined to be regulated by ET and/or MeJA via pairwise comparisons among the three exposure durations (0 h, 6 h, and 48 h). Proteins associated with latex regeneration, including phosphoenolpyruvate carboxylase and acetyl-CoA C-acetyltransferase, and those associated with latex flow, such as chitinase and a sieve element occlusion protein, were affected by the application of ET. Chitinase and polyphenol oxidase were also found to be regulated by MeJA. The findings of this study may provide new insight into the roles of phytohormones in latex yield and the causative mechanisms of laticifer differentiation in rubber trees. On the basis of a transcriptome shotgun assembly (TSA) sequence database and an iTRAQ-coupled two-dimensional LC-MS/MS approach, the most comprehensive proteome of the latex was profiled, and the ethylene-/jasmonate-responsive proteins were identified in the latex of H. brasiliensis. The findings of this study may provide new insight into the role of phytohormones in latex yield and the causative mechanisms of laticifer differentiation in rubber trees. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Critical assessment of alignment procedures for LC-MS proteomics and metabolomics measurements

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    Neumann Steffen


    Full Text Available Abstract Background Liquid chromatography coupled to mass spectrometry (LC-MS has become a prominent tool for the analysis of complex proteomics and metabolomics samples. In many applications multiple LC-MS measurements need to be compared, e. g. to improve reliability or to combine results from different samples in a statistical comparative analysis. As in all physical experiments, LC-MS data are affected by uncertainties, and variability of retention time is encountered in all data sets. It is therefore necessary to estimate and correct the underlying distortions of the retention time axis to search for corresponding compounds in different samples. To this end, a variety of so-called LC-MS map alignment algorithms have been developed during the last four years. Most of these approaches are well documented, but they are usually evaluated on very specific samples only. So far, no publication has been assessing different alignment algorithms using a standard LC-MS sample along with commonly used quality criteria. Results We propose two LC-MS proteomics as well as two LC-MS metabolomics data sets that represent typical alignment scenarios. Furthermore, we introduce a new quality measure for the evaluation of LC-MS alignment algorithms. Using the four data sets to compare six freely available alignment algorithms proposed for the alignment of metabolomics and proteomics LC-MS measurements, we found significant differences with respect to alignment quality, running time, and usability in general. Conclusion The multitude of available alignment methods necessitates the generation of standard data sets and quality measures that allow users as well as developers to benchmark and compare their map alignment tools on a fair basis. Our study represents a first step in this direction. Currently, the installation and evaluation of the "correct" parameter settings can be quite a time-consuming task, and the success of a particular method is still highly

  5. The APEX Quantitative Proteomics Tool: Generating protein quantitation estimates from LC-MS/MS proteomics results

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    Saeed Alexander I


    utility to merge multiple APEX results into a standardized format in preparation for further statistical analysis. Conclusion The APEX Quantitative Proteomics Tool provides a simple means to quickly derive hundreds to thousands of protein abundance values from standard liquid chromatography-tandem mass spectrometry proteomics datasets. The APEX tool provides a straightforward intuitive interface design overlaying a highly customizable computational workflow to produce protein abundance values from LC-MS/MS datasets.

  6. Comparative proteomic exploration of whey proteins in human and bovine colostrum and mature milk using iTRAQ-coupled LC-MS/MS. (United States)

    Yang, Mei; Cao, Xueyan; Wu, Rina; Liu, Biao; Ye, Wenhui; Yue, Xiqing; Wu, Junrui


    Whey, an essential source of dietary nutrients, is widely used in dairy foods for infants. A total of 584 whey proteins in human and bovine colostrum and mature milk were identified and quantified by the isobaric tag for relative and absolute quantification (iTRAQ) proteomic method. The 424 differentially expressed whey proteins were identified and analyzed according to gene ontology (GO) annotation, Kyoto encyclopedia of genes and genomes (KEGG) pathway, and multivariate statistical analysis. Biological processes principally involved biological regulation and response to stimulus. Major cellular components were extracellular region part and extracellular space. The most prevalent molecular function was protein binding. Twenty immune-related proteins and 13 proteins related to enzyme regulatory activity were differentially expressed in human and bovine milk. Differentially expressed whey proteins participated in many KEGG pathways, including major complement and coagulation cascades and in phagosomes. Whey proteins show obvious differences in expression in human and bovine colostrum and mature milk, with consequences for biological function. The results here increase our understanding of different whey proteomes, which could provide useful information for the development and manufacture of dairy products and nutrient food for infants. The advanced iTRAQ proteomic approach was used to analyze differentially expressed whey proteins in human and bovine colostrum and mature milk.

  7. Application of survival analysis methodology to the quantitative analysis of LC-MS proteomics data

    KAUST Repository

    Tekwe, C. D.


    MOTIVATION: Protein abundance in quantitative proteomics is often based on observed spectral features derived from liquid chromatography mass spectrometry (LC-MS) or LC-MS/MS experiments. Peak intensities are largely non-normal in distribution. Furthermore, LC-MS-based proteomics data frequently have large proportions of missing peak intensities due to censoring mechanisms on low-abundance spectral features. Recognizing that the observed peak intensities detected with the LC-MS method are all positive, skewed and often left-censored, we propose using survival methodology to carry out differential expression analysis of proteins. Various standard statistical techniques including non-parametric tests such as the Kolmogorov-Smirnov and Wilcoxon-Mann-Whitney rank sum tests, and the parametric survival model and accelerated failure time-model with log-normal, log-logistic and Weibull distributions were used to detect any differentially expressed proteins. The statistical operating characteristics of each method are explored using both real and simulated datasets. RESULTS: Survival methods generally have greater statistical power than standard differential expression methods when the proportion of missing protein level data is 5% or more. In particular, the AFT models we consider consistently achieve greater statistical power than standard testing procedures, with the discrepancy widening with increasing missingness in the proportions. AVAILABILITY: The testing procedures discussed in this article can all be performed using readily available software such as R. The R codes are provided as supplemental materials. CONTACT:

  8. Hydrophilic interaction chromatography using a meter-scale monolithic silica capillary column for proteomics LC-MS. (United States)

    Horie, Kanta; Kamakura, Takeo; Ikegami, Tohru; Wakabayashi, Masaki; Kato, Takashi; Tanaka, Nobuo; Ishihama, Yasushi


    A meter-scale monolithic silica capillary column modified with urea-functional groups for hydrophilic interaction liquid chromatography (HILIC) was developed for highly efficient separation of biological compounds. We prepared a ureidopropylsilylated monolithic silica capillary column with a minimum plate height of 12 μm for nucleosides and a permeability of 2.1 × 10(-13) m(2), which is comparable with the parameters of monolithic silica-C18 capillary columns. Over 300,000 theoretical plates were experimentally obtained in HILIC with a 4 m long column at 8 MPa; this is the best result yet reported for HILIC. A 2 m long ureidopropylsilylated monolithic silica capillary column was utilized to develop a HILIC mode LC-MS system for proteomics applications. Using tryptic peptides from human HeLa cell lysate proteins, we identified the comparable numbers of peptides and proteins in HILIC with those in reversed-phase liquid chromatography (RPLC) using a C18-modified monolithic silica column when shallow gradients were applied. In addition, approximately 5-fold increase in the peak response on average was observed in HILIC for commonly identified tryptic peptides due to the high acetonitrile concentration in the HILIC mobile phase. Since HILIC mode LC-MS shows orthogonal selectivity to RPLC mode LC-MS, it is useful as a complementary tool to increase proteome coverage in proteomics studies.

  9. QuaMeter: multivendor performance metrics for LC-MS/MS proteomics instrumentation. (United States)

    Ma, Ze-Qiang; Polzin, Kenneth O; Dasari, Surendra; Chambers, Matthew C; Schilling, Birgit; Gibson, Bradford W; Tran, Bao Q; Vega-Montoto, Lorenzo; Liebler, Daniel C; Tabb, David L


    LC-MS/MS-based proteomics studies rely on stable analytical system performance that can be evaluated by objective criteria. The National Institute of Standards and Technology (NIST) introduced the MSQC software to compute diverse metrics from experimental LC-MS/MS data, enabling quality analysis and quality control (QA/QC) of proteomics instrumentation. In practice, however, several attributes of the MSQC software prevent its use for routine instrument monitoring. Here, we present QuaMeter, an open-source tool that improves MSQC in several aspects. QuaMeter can directly read raw data from instruments manufactured by different vendors. The software can work with a wide variety of peptide identification software for improved reliability and flexibility. Finally, QC metrics implemented in QuaMeter are rigorously defined and tested. The source code and binary versions of QuaMeter are available under Apache 2.0 License at

  10. GeLC-MS: A Sample Preparation Method for Proteomics Analysis of Minimal Amount of Tissue. (United States)

    Makridakis, Manousos; Vlahou, Antonia


    Application of various proteomics methodologies have been implemented for the global and targeted proteome analysis of many different types of biological samples such as tissue, urine, plasma, serum, blood, and cell lines. Among the aforementioned biological samples, tissue has an exceptional role into clinical research and practice. Disease initiation and progression is usually located at the tissue level of different organs, making the analysis of this material very important for the understanding of the disease pathophysiology. Despite the significant advances in the mass spectrometry instrumentation, tissue proteomics still faces several challenges mainly due to increased sample complexity and heterogeneity. However, the most prominent challenge is attributed to the invasive procedure of tissue sampling which restricts the availability of fresh frozen tissue to minimal amounts and limited number of samples. Application of GeLC-MS sample preparation protocol for tissue proteomics analysis can greatly facilitate making up for these difficulties. In this chapter, a step by step guide for the proteomics analysis of minute amounts of tissue samples using the GeLC-MS sample preparation protocol, as applied by our group in the analysis of multiple different types of tissues (vessels, kidney, bladder, prostate, heart) is provided.

  11. MASPECTRAS: a platform for management and analysis of proteomics LC-MS/MS data

    Directory of Open Access Journals (Sweden)

    Rader Robert


    Full Text Available Abstract Background The advancements of proteomics technologies have led to a rapid increase in the number, size and rate at which datasets are generated. Managing and extracting valuable information from such datasets requires the use of data management platforms and computational approaches. Results We have developed the MAss SPECTRometry Analysis System (MASPECTRAS, a platform for management and analysis of proteomics LC-MS/MS data. MASPECTRAS is based on the Proteome Experimental Data Repository (PEDRo relational database schema and follows the guidelines of the Proteomics Standards Initiative (PSI. Analysis modules include: 1 import and parsing of the results from the search engines SEQUEST, Mascot, Spectrum Mill, X! Tandem, and OMSSA; 2 peptide validation, 3 clustering of proteins based on Markov Clustering and multiple alignments; and 4 quantification using the Automated Statistical Analysis of Protein Abundance Ratios algorithm (ASAPRatio. The system provides customizable data retrieval and visualization tools, as well as export to PRoteomics IDEntifications public repository (PRIDE. MASPECTRAS is freely available at Conclusion Given the unique features and the flexibility due to the use of standard software technology, our platform represents significant advance and could be of great interest to the proteomics community.

  12. MET-XAlign: a metabolite cross-alignment tool for LC/MS-based comparative metabolomics. (United States)

    Zhang, Wenchao; Lei, Zhentian; Huhman, David; Sumner, Lloyd W; Zhao, Patrick X


    Liquid chromatography/mass spectrometry (LC/MS) metabolite profiling has been widely used in comparative metabolomics studies; however, LC/MS-based comparative metabolomics currently faces several critical challenges. One of the greatest challenges is how to effectively align metabolites across different LC/MS profiles; a single metabolite can give rise to multiple peak features, and the grouped peak features that can be used to construct a spectrum pattern of single metabolite can vary greatly between biochemical experiments and even between instrument runs. Another major challenge is that the observed retention time for a single metabolite can also be significantly affected by experimental conditions. To overcome these two key challenges, we present a novel metabolite-based alignment approach entitled MET-XAlign to align metabolites across LC/MS metabolomics profiles. MET-XAlign takes the deduced molecular mass and estimated compound retention time information that can be extracted by our previously published tool, MET-COFEA, and aligns metabolites based on this information. We demonstrate that MET-XAlign is able to cross-align metabolite compounds, either known or unknown, in LC/MS profiles not only across different samples but also across different biological experiments and different electrospray ionization modes. Therefore, our proposed metabolite-based cross-alignment approach is a great step forward and its implementation, MET-XAlign, is a very useful tool in LC/MS-based comparative metabolomics. MET-XAlign has been successfully implemented with core algorithm coding in C++, making it very efficient, and visualization interface coding in the Microsoft.NET Framework. The MET-XAlign software along with demonstrative data is freely available at .

  13. Plasma Membrane Proteomics of Arabidopsis Suspension-Cultured Cells Associated with Growth Phase Using Nano-LC-MS/MS. (United States)

    Li, Bin; Takahashi, Daisuke; Kawamura, Yukio; Uemura, Matsuo


    Arabidopsis thaliana suspension-cultured cells (T87 line) are important model system for studies of responses to biotic and abiotic stresses at the cellular level in vitro since the cells have certain advantages compared with the whole plant system. However, the physiological and morphological characteristics of the cells are influenced by the progress of the growth phase of cells, which may result in different stress tolerance. To obtain comprehensive proteome profiles of the plasma membrane of Arabidopsis thaliana T87 suspension-cultured cells at the lag, log, or stationary growth phase, a shotgun proteomics method using nano-LC-MS/MS is used. The results obtained indicate that proteome profiles of the plasma membrane with the progress of the growth phase of cells dynamically changed, which may be associated with the physiological and morphological characteristics of the plasma membrane of the suspension-cultured cells. The proteomics results are further applied to explain different responsive patterns in the plasma membrane to cold acclimation and ABA treatment, which lead to understanding of different freezing tolerance associated with the growth phase of the cells.

  14. A comprehensive full factorial LC-MS/MS proteomics benchmark data set.

    NARCIS (Netherlands)

    Wessels, H.J.C.T.; Bloemberg, T.G.; Dael, M.F.P. van; Wehrens, R.; Buydens, L.M.; Heuvel, L.P.W.J. van den; Gloerich, J.


    An important prerequisite for the development and benchmarking of novel analysis methods is a well-designed comprehensive LC-MS/MS data set. Here, we present our data set consisting of 59 LC-MS/MS analyses of 50 protein samples extracted individually from Escherichia coli K12 and spiked with

  15. Mass spectrometry (LC-MS/MS) identified proteomic biosignatures of breast cancer in proximal fluid (United States)

    Whelan, Stephen A.; He, Jianbo; Lu, Ming; Souda, Puneet; Saxton, Romaine E.; Faull, Kym F.; Whitelegge, Julian P.; Chang, Helena R.


    Summary We have begun an early phase of biomarker discovery in three clinically important types of breast cancer using a panel of human cell lines: HER2 positive, HER2 negative and hormone receptor positive and triple negative (HER2−, ER−, PR−). We identified and characterized the most abundant secreted, sloughed, or leaked proteins released into serum free media from these breast cancer cell lines using a combination of protein fractionation methods before LC-MS/MS mass spectrometry analysis. A total of 249 proteins were detected in the proximal fluid of 7 breast cancer cell lines. The expression of a selected group of high abundance and/or breast cancer specific potential biomarkers including thromobospondin 1, galectin-3 binding protein, cathepsin D, vimentin, zinc-α2-glycoprotein, CD44, and EGFR from the breast cancer cell lines and in their culture media were further validated by Western blot analysis. Interestingly, mass spectrometry identified a cathepsin D protein single-nucleotide polymorphism (SNP) by alanine to valine replacement from the MCF-7 breast cancer cell line. Comparison of each cell line media proteome displayed unique and consistent biosignatures regardless of the individual group classifications demonstrating the potential for stratification of breast cancer. Based on the cell line media proteome, predictive Tree software was able to categorize each cell line as HER2 positive, HER2 negative and hormone receptor positive and triple negative based on only two proteins, muscle fructose 1,6-bisphosphate aldolase and keratin 19. In addition, the predictive Tree software clearly identified MCF-7 cell line overexpresing the HER2 receptor with the SNP cathepsin D biomarker. PMID:22934887

  16. Comparison of bottom-up proteomic approaches for LC-MS analysis of complex proteomes. (United States)

    Weston, Leigh A; Bauer, Kerry M; Hummon, Amanda B


    Discovery-based proteomic studies aim to answer important biological questions by identifying as many proteins as possible. In order to accomplish this lofty goal, an effort must be placed on determining an optimal workflow that maximizes protein identifications. In this study, we compare protein extraction, digestion and fractionation methods for bottom-up proteomics using a human colon cancer cell line as our model system. Four different buffers for protein extraction, two digestion approaches, as well as three sample fractionation methods were evaluated in order to determine an accessible workflow that gives maximal protein identifications. Samples comparing these workflows were analyzed via UPLC paired with tandem MS on a Q-Exactive mass spectrometer. Our goal is to determine an optimal workflow to enable users to maximize protein identifications. Our results show that an increased number of confident protein identifications are attained with a filter-aided digestion approach as compared to an in-solution digestion. Overall SDS-PAGE fractionation leads to higher numbers of identifications than SCX SpinTip and reverse phased cartridge platforms. The novel aspect of this work is the comparison of two readily available, offline platforms for fractionation in reference to a traditional technique, SDS-PAGE.

  17. Quantitative proteomic analysis of Huh-7 cells infected with Dengue virus by label-free LC-MS. (United States)

    Pando-Robles, Victoria; Oses-Prieto, Juan A; Rodríguez-Gandarilla, Myriam; Meneses-Romero, Erika; Burlingame, Alma L; Batista, Cesar V F


    Dengue is an important and growing public health problem worldwide with an estimated 100million new clinical cases annually. Currently, no licensed drug or vaccine is available. During natural infection in humans, liver cells constitute one of the main targets of dengue virus (DENV) replication. However, a clear understanding of dengue pathogenesis remains elusive. In order to gain a better reading of the cross talk between virus and host cell proteins, we used a proteomics approach to analyze the host response to DENV infection in a hepatic cell line Huh-7. Differences in proteome expression were assayed 24h post-infection using label-free LC-MS. Quantitative analysis revealed 155 differentially expressed proteins, 64 of which were up-regulated and 91 down-regulated. These results reveal an important decrease in the expression of enzymes involved in the glycolytic pathway, citrate cycle, and pyruvate metabolism. This study provides large-scale quantitative information regarding protein expression in the early stages of infection that should be useful for better compression of the pathogenesis of dengue. Dengue infection involves alterations in the homeostasis of the host cell. Defining the interactions between virus and cell proteins should provide a better understanding of how viruses propagate and cause disease. Here, we present for the first time the proteomic analysis of hepatocytes (Huh-7 cells) infected with DENV-2 by label-free LC-MS. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Mapping the Subcellular Proteome of Shewanella oneidensis MR-1 using Sarkosyl-based fractionation and LC-MS/MS protein identification

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    Brown, Roslyn N.; Romine, Margaret F.; Schepmoes, Athena A.; Smith, Richard D.; Lipton, Mary S.


    A simple and effective subcellular proteomic method for fractionation and analysis of gram-negative bacterial cytoplasm, periplasm, inner, and outer membranes was applied to Shewanella oneidensis to gain insight into its subcellular architecture. A combination of differential centrifugation, Sarkosyl solubilization, and osmotic lysis was used to prepare subcellular fractions. Global differences in protein fractions were observed by SDS PAGE and heme staining, and tryptic peptides were analyzed using high-resolution LC-MS/MS. Compared to crude cell lysates, the fractionation method achieved a significant enrichment (average ~2-fold) in proteins predicted to be localized to each subcellular fraction. Compared to other detergent, organic solvent, and density-based methods previously reported, Sarkosyl most effectively facilitated separation of the inner and outer membranes and was amenable to mass spectrometry, making this procedure ideal for probing the subcellular proteome of gram-negative bacteria via LC-MS/MS. With 40% of the observable proteome represented, this study has provided extensive information on both subcellular architecture and relative abundance of proteins in S. oneidensis and provides a foundation for future work on subcellular organization and protein-membrane interactions in other gram-negative bacteria.

  19. Short Communication: Testosterone Measured with an Automatic Immunoassay Compares Reasonbly Well to Results Obtained by LC-MS/MS

    DEFF Research Database (Denmark)

    Knudsen, Cindy Søndersø; Højskov, Carsten Schriver; Møller, Holger Jon


    Background: Previous studies have reported problems measuring testosterone with immunological assays. Here we explore an automatic second generation immunoassay compared to a LC-MS/MS method. Methods: We collected blood samples from 76 women and measured testosterone, progesterone, gender...... hormonebinding globulin (SHBG), and albumin employing Cobas e601/c501. Testosterone, androstenedione (andro), dehydroepiandrosterone sulphate (DHEAS), and 17-hydroxyprogesterone (17-OHP) concentrations were measured employing LC-MS/MS. We evaluated the difference between testosterone measured by the two methods...... for the difference between results obtained by the two methods and the sample concentration of DHEAS and andro: Diff (Cobas e601 - LC-MS/MS) = 0.116 x DHEAS - 0.396, r = 0.84 and Diff (Cobas e601 - LC-MS/MS) = 0.08 andro - 0.380, r = 0.58. No statistically significant interference was observed for progesterone, 17...

  20. LC-MS/MS analysis of visceral and subcutaneous adipose tissue proteomes in young goats with focus on innate immunity and inflammation related proteins

    DEFF Research Database (Denmark)

    Restelli, Laura; Codrea, Marius Cosmin; Savoini, Giovanni


    and visceral adipose tissues of goat, focusing on proteins involved in immune and inflammatory response. A 2-D LC-MS/MS approach followed by cluster analysis shows a clear distinction between subcutaneous and visceral fat tissue proteomes, and qualitative RT-PCR based analysis of 30 potential adipokines...

  1. Mycotoxin Analysis of Human Urine by LC-MS/MS: A Comparative Extraction Study (United States)

    Escrivá, Laura; Font, Guillermina


    The lower mycotoxin levels detected in urine make the development of sensitive and accurate analytical methods essential. Three extraction methods, namely salting-out liquid–liquid extraction (SALLE), miniQuEChERS (quick, easy, cheap, effective, rugged, and safe), and dispersive liquid–liquid microextraction (DLLME), were evaluated and compared based on analytical parameters for the quantitative LC-MS/MS measurement of 11 mycotoxins (AFB1, AFB2, AFG1, AFG2, OTA, ZEA, BEA, EN A, EN B, EN A1 and EN B1) in human urine. DLLME was selected as the most appropriate methodology, as it produced better validation results for recovery (79–113%), reproducibility (RSDs < 12%), and repeatability (RSDs < 15%) than miniQuEChERS (71–109%, RSDs <14% and <24%, respectively) and SALLE (70–108%, RSDs < 14% and < 24%, respectively). Moreover, the lowest detection (LODS) and quantitation limits (LOQS) were achieved with DLLME (LODs: 0.005–2 μg L−1, LOQs: 0.1–4 μg L−1). DLLME methodology was used for the analysis of 10 real urine samples from healthy volunteers showing the presence of ENs B, B1 and A1 at low concentrations. PMID:29048356

  2. Implementation of Statistical Process Control for Proteomic Experiments Via LC MS/MS (United States)

    Bereman, Michael S.; Johnson, Richard; Bollinger, James; Boss, Yuval; Shulman, Nick; MacLean, Brendan; Hoofnagle, Andrew N.; MacCoss, Michael J.


    Statistical process control (SPC) is a robust set of tools that aids in the visualization, detection, and identification of assignable causes of variation in any process that creates products, services, or information. A tool has been developed termed Statistical Process Control in Proteomics (SProCoP) which implements aspects of SPC (e.g., control charts and Pareto analysis) into the Skyline proteomics software. It monitors five quality control metrics in a shotgun or targeted proteomic workflow. None of these metrics require peptide identification. The source code, written in the R statistical language, runs directly from the Skyline interface, which supports the use of raw data files from several of the mass spectrometry vendors. It provides real time evaluation of the chromatographic performance (e.g., retention time reproducibility, peak asymmetry, and resolution), and mass spectrometric performance (targeted peptide ion intensity and mass measurement accuracy for high resolving power instruments) via control charts. Thresholds are experiment- and instrument-specific and are determined empirically from user-defined quality control standards that enable the separation of random noise and systematic error. Finally, Pareto analysis provides a summary of performance metrics and guides the user to metrics with high variance. The utility of these charts to evaluate proteomic experiments is illustrated in two case studies.

  3. Generation of accurate peptide retention data for targeted and data independent quantitative LC-MS analysis: Chromatographic lessons in proteomics. (United States)

    Krokhin, Oleg V; Spicer, Vic


    The emergence of data-independent quantitative LC-MS/MS analysis protocols further highlights the importance of high-quality reproducible chromatographic procedures. Knowing, controlling and being able to predict the effect of multiple factors that alter peptide RP-HPLC separation selectivity is critical for successful data collection for the construction of ion libraries. Proteomic researchers have often regarded RP-HPLC as a "black box", while vast amount of research on peptide separation is readily available. In addition to obvious parameters, such as the type of ion-pairing modifier, stationary phase and column temperature, we describe the "mysterious" effects of gradient slope, column size and flow rate on peptide separation selectivity. Retention time variations due to these parameters are governed by the linear solvent strength (LSS) theory on a peptide level by the value of its slope S in the basic LSS equation-a parameter that can be accurately predicted. Thus, the application of shallower gradients, higher flow rates, or smaller columns will each increases the relative retention of peptides with higher S-values (long species with multiple positively charged groups). Simultaneous changes to these parameters that each drive shifts in separation selectivity in the same direction should be avoided. The unification of terminology represents another pressing issue in this field of applied proteomics that should be addressed to facilitate further progress. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Part I: Characterization of the Extracellular Proteome of the Extreme Thermophile Caldicellulosiruptor saccharolyticus by GeLC-MS2 (United States)

    Muddiman, David; Andrews, Genna; Lewis, Derrick; Notey, Jaspreet; Kelly, Robert


    The proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. The secretome, or extracellular proteome of these microorganisms no doubt harbors technologically important enzymes and other thermostable biomolecules that to date have been characterized only to a limited extent. In the first of a two part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negative impact on all downstream experiments. Following efficient secretome purification, GeLC-MS2 analysis and prediction servers suggest probable protein secretion to complement experimental data. In an effort to define the extracellular proteome of the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus, several techniques were considered regarding sample processing to achieve the most in-depth analysis of secreted proteins. Order of operation experiments all including the C18 bead technique demonstrated that two levels of sample purification were necessary to effectively de-salt the sample and provide sufficient protein identifications. Five sample preparation combinations yielded 71 proteins and the majority described as enzymatic and putative uncharacterized proteins anticipating consolidated bioprocessing applications. Nineteen proteins were predicted by Phobius, SignalP, SecretomeP, or TatP for extracellular secretion and 11 contain transmembrane domain stretches suggested by Phobius and TMHMM. The sample preparation technique demonstrating the most effective outcome for C. saccharolyticus secreted proteins in this study involves acetone precipitation followed by the C18 bead method in which 2.4% (63 proteins) of the predicted proteome was indentified including proteins suggested to have secretion and transmembrane moieties. PMID:20623222

  5. Discovery of novel glucose-regulated proteins in isolated human pancreatic islets using LC-MS/MS-based proteomics

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    Rutledge, Alexandra C.; Fontes, Ghislaine; Gritsenko, Marina A.; Norbeck, Angela D.; Anderson, David J.; Waters, Katrina M.; Adkins, Joshua N.; Smith, Richard D.; Poitout, Vincent; Metz, Thomas O.


    The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is due in part to insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent and physiologically important regulators of beta-cell function under physiological conditions, understanding the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins ({approx}p < 0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (Pleiotropic regulator 1), processing (Retinoblastoma binding protein 6), and function (Nuclear RNA export factor 1), in addition to Neuron navigator 1 and Plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and Synaptotagmin-17. Many proteins found to be differentially abundant after high glucose stimulation were uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.

  6. Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset. (United States)

    Ramus, Claire; Hovasse, Agnès; Marcellin, Marlène; Hesse, Anne-Marie; Mouton-Barbosa, Emmanuelle; Bouyssié, David; Vaca, Sebastian; Carapito, Christine; Chaoui, Karima; Bruley, Christophe; Garin, Jérôme; Cianférani, Sarah; Ferro, Myriam; Van Dorssaeler, Alain; Burlet-Schiltz, Odile; Schaeffer, Christine; Couté, Yohann; Gonzalez de Peredo, Anne


    Proteomic workflows based on nanoLC-MS/MS data-dependent-acquisition analysis have progressed tremendously in recent years. High-resolution and fast sequencing instruments have enabled the use of label-free quantitative methods, based either on spectral counting or on MS signal analysis, which appear as an attractive way to analyze differential protein expression in complex biological samples. However, the computational processing of the data for label-free quantification still remains a challenge. Here, we used a proteomic standard composed of an equimolar mixture of 48 human proteins (Sigma UPS1) spiked at different concentrations into a background of yeast cell lysate to benchmark several label-free quantitative workflows, involving different software packages developed in recent years. This experimental design allowed to finely assess their performances in terms of sensitivity and false discovery rate, by measuring the number of true and false-positive (respectively UPS1 or yeast background proteins found as differential). The spiked standard dataset has been deposited to the ProteomeXchange repository with the identifier PXD001819 and can be used to benchmark other label-free workflows, adjust software parameter settings, improve algorithms for extraction of the quantitative metrics from raw MS data, or evaluate downstream statistical methods. Bioinformatic pipelines for label-free quantitative analysis must be objectively evaluated in their ability to detect variant proteins with good sensitivity and low false discovery rate in large-scale proteomic studies. This can be done through the use of complex spiked samples, for which the "ground truth" of variant proteins is known, allowing a statistical evaluation of the performances of the data processing workflow. We provide here such a controlled standard dataset and used it to evaluate the performances of several label-free bioinformatics tools (including MaxQuant, Skyline, MFPaQ, IRMa-hEIDI and Scaffold) in

  7. [Development and application of FD-LC-MS/MS proteomics analysis revealing protein expression and biochemical events in tissues and cells]. (United States)

    Ichibangase, Tomoko; Imai, Kazuhiro


    It is routine to search for and recognized genetic defects in human disorders to provide knowledge for diagnosis, treatment, and protection against diseases. It is also important to investigate and demonstrate the cause of a disease from the proteomic perspective, because intracellular signaling systems depend on protein dynamics. Demonstrating changes in protein levels enables us to understand biochemical events during the initiation and progression of a disease. To understand changes in protein levels in tissues and cells, we have developed a novel proteomics approach, FD-LC-MS/ MS. This consists of fluorogenic derivatization (FD), HPLC separation and detection/quantification of proteins in a biological sample, followed by the isolation and tryptic digestion of target proteins, and then their identification using HPLC and tandem mass spectrometry (MS/MS) with a database-searching algorithm. The method is highly sensitive (femtomole-level detection) through the use of less noisy fluorogenic rather than fluorescence derivatization, and enables precise and comprehensive relative quantitation of protein levels (between-day relative standard deviation of peak heights of ca. 20%) by combining FD with HPLC separation. In this paper, after a simple review of differential profiling using FD-LC-MS/MS, for example the analysis of stimulated vs. unstimulated samples, we introduce the development and application of the FD-LC-MS/MS method for comprehensive differential proteomics of several tissues, including mouse liver, mouse brain, and breast cancer cell lines, to reveal protein levels and biochemical events in tissues and cells.

  8. Data processing pipelines for comprehensive profiling of proteomics samples by label-free LC MS for biomarker discovery

    NARCIS (Netherlands)

    Christin, Christin; Bischoff, Rainer; Horvatovich, Peter


    Label-free quantitative LC-MS profiling of complex body fluids has become an important analytical tool for biomarker and biological knowledge discovery in the past decade. Accurate processing, statistical analysis and validation of acquired data diversified by the different types of mass

  9. Analysis of membrane proteome by data-dependent LC-MS/MS combined with data-independent LC-MSE technique

    Directory of Open Access Journals (Sweden)

    Joseph Kwon


    Full Text Available Proteomics work resembles the search for a needle in a haystack. The identification of protein biomarker requires the removal of the false protein data from the whole protein mixture. For high quality proteomic data, even a strict filtration step using the false discovery rate (FDR is insufficient for obtaining perfect protein information from the biological samples. In this study, the cyanobacterial whole membrane fraction was applied to the data-dependent analysis (DDA mode of LC-MS/MS, which was used along with the data-independent LC-MSE technique in order to evaluate the membrane proteomic data. Furthermore, the identified MSE-information (MSE-i data based on the peptide mass and the retention time were validated by the other database search, i.e., the probability-based MASCOT and de novo search engine PEAKS. In this present study, 208 cyanobacterial proteins with FDR of 5% were identified using the data-independent nano-UPLC/MSE acquisition with the Protein Lynx Global Server (PLGS, and 56 of these proteins were the predicted membrane proteins. When a total of 208 MSE-i proteomic data were applied to the DDA mode of LC-MS/MS, the number of identified membrane proteins was 26 and 33 from MASCOT and PEAKS with a FDR of 5%, respectively. The number of totally overlapped membrane proteins was 25. Therefore, the data-independent LC-MSE identified more proteins with a high confidence.

  10. Development of an Efficient Protein Extraction Method Compatible with LC-MS/MS for Proteome Mapping in Two Australian Seagrasses Zostera muelleri and Posidonia australis

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    Zhijian Jiang


    Full Text Available The availability of the first complete genome sequence of the marine flowering plant Zostera marina (commonly known as seagrass in early 2016, is expected to significantly raise the impact of seagrass proteomics. Seagrasses are marine ecosystem engineers that are currently declining worldwide at an alarming rate due to both natural and anthropogenic disturbances. Seagrasses (especially species of the genus Zostera are compromised for proteomic studies primarily due to the lack of efficient protein extraction methods because of their recalcitrant cell wall which is rich in complex polysaccharides and a high abundance of secondary metabolites in their cells. In the present study, three protein extraction methods that are commonly used in plant proteomics i.e., phenol (P; trichloroacetic acid/acetone/SDS/phenol (TASP; and borax/polyvinyl-polypyrrolidone/phenol (BPP extraction, were evaluated quantitatively and qualitatively based on two dimensional isoelectric focusing (2D-IEF maps and LC-MS/MS analysis using the two most abundant Australian seagrass species, namely Zostera muelleri and Posidonia australis. All three tested methods produced high quality protein extracts with excellent 2D-IEF maps in P. australis. However, the BPP method produces better results in Z. muelleri compared to TASP and P. Therefore, we further modified the BPP method (M-BPP by homogenizing the tissue in a modified protein extraction buffer containing both ionic and non-ionic detergents (0.5% SDS; 1.5% Triton X-100, 2% PVPP and protease inhibitors. Further, the extracted proteins were solubilized in 0.5% of zwitterionic detergent (C7BzO instead of 4% CHAPS. This slight modification to the BPP method resulted in a higher protein yield, and good quality 2-DE maps with a higher number of protein spots in both the tested seagrasses. Further, the M-BPP method was successfully utilized in western-blot analysis of phosphoenolpyruvate carboxylase (PEPC—a key enzyme for carbon

  11. Ultrasensitive Proteome Profiling for 100 Living Cells by Direct Cell Injection, Online Digestion and Nano-LC-MS/MS Analysis. (United States)

    Chen, Qi; Yan, Guoquan; Gao, Mingxia; Zhang, Xiangmin


    Single-cell proteome analysis has always been an exciting goal because it provides crucial information about cellular heterogeneity and dynamic change. Here we presented an integrated proteome analysis device (iPAD) for 100 living cells (iPAD-100) that might be suitable for single-cell analysis. Once cells were cultured, the iPAD-100 could be applied to inject 100 living cells, to transform the living cells into peptides, and to produce protein identification results with total automation. Due to the major obstacle for detection limit of mass spectrometry, we applied the iPAD-100 to analyze the proteome of 100 cells. In total, 813 proteins were identified in a DLD-cell proteome by three duplicate runs. Gene Ontology analysis revealed that proteins from different cellular compartments were well-represented, including membrane proteins. The iPAD-100 greatly simplified the sampling process, reduced sample loss, and prevented contamination. As a result, proteins whose copy numbers were lower than 1000 were identified from 100-cell samples with the iPAD-100, showing that a detection limit of 200 zmol was achieved. With increased sensitivity of mass spectrometry, the iPAD-100 may be able to reveal bountiful proteome information from a single cell in the near future.

  12. LC-MS Proteomics Analysis of the Insulin/IGF-1 Deficient Caenorhabditis elegans daf-2(e1370) Mutant Reveals Extensive Restructuring of Intermediary Metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Depuydt, Geert G.; Xie, Fang; Petyuk, Vladislav A.; Smolders, Arne; Brewer, Heather M.; Camp, David G.; Smith, Richard D.; Braeckman, Bart P.


    The insulin/IGF-1 receptor is a major known determinant of dauer formation, stress resistance, longevity and metabolism in C. elegans. In the past, whole-genome transcript profiling was used extensively to study differential gene expression in response to reduced insulin/IGF-1 signaling, including expression levels of metabolism-associated genes. Taking advantage of the recent developments in quantitative liquid chromatography mass-spectrometry (LC-MS) based proteomics, we profiled the proteomic changes that occur in response to activation of the DAF-16 transcription factor in the germline-less glp-4(bn2); daf-2(e1370) receptor mutant. Strikingly, the daf-2 profile suggests extensive reorganization of intermediary metabolism, characterized by the up-regulation of many core intermediary metabolic pathways. These include, glycolysis/gluconeogenesis, glycogenesis, pentose phosphate cycle, citric acid cycle, glyoxylate shunt, fatty acid β-oxidation, one-carbon metabolism, propionate and tyrosine catabolism, and complex I, II, III and V of the electron transport chain. Interestingly, we found simultaneous activation of reciprocally regulated metabolic pathways, which is indicative for spatio-temporal coordination of energy metabolism and/or extensive post-translational regulation of these enzymes. This restructuring of daf-2 metabolism is reminiscent to that of hypometabolic dauers, allowing the efficient and economical utilization of internal nutrient reserves, possibly also shunting metabolites through alternative energy-generating pathways, in order to sustain longevity.

  13. Comparative Analysis of the Bufonis Venenum by Using TLC, HPLC, and LC-MS for Different Extraction Methods. (United States)

    Lee, Hyo-Jae; Koung, Fan-Pei; Kwon, Ki-Rok; Kang, Dae-In; Cohen, Lorenzo; Yang, Pei-Ying; Yoo, Hwa-Seung


    Toad venom, called Chan-Su, is a traditional Oriental medicine secreted from the auricular and the skin glands of the Bufo bufo gargarizanz Cantor or B. melanosticus Schneider and has been widely used in China, Korea and other parts of Asia for the treatment of pain, heart conditions, and cancer. We examined the concentrations of the main chemical constituents within a commerciallyavailable toad venom product and compared the levels for different extraction methods. Toad venom was extracted using either cold or hot water, ethanol (EtOH), methanol (MeOH), or ethyl acetate (EtOAc), was fractionated using precipitation or reflux, and was then analyzed using thin layer chromatography (TLC), high-performance liquid chromatography (HTLC), and liquid chroma-tography - mass spectrometry (LC-MS). Individual components were identified by comparisons of the retention times, the ultraviolet spectra, and mass spectras and differences in chemical constituents for different solvents and extraction methods are presented. Components with authentic standards, including serotonin and bufodienolides (cinobufagen, bufalin, cinobufalin, and resibufogenin), were detected. The water extract of toad venom contained the greatest amount of serotonin (75.7 ± 0.1 mg/g), but very small amounts of bufodienolides (3.8 ± 0.0 mg/g). In contrast, the use of MeOH or EtOH extraction solutions resulted in 5-26 times higher concentrations of bufodienolides, with only trace amounts of serotonin. The relative and the absolute concentrations of the component also varied based on the extraction method; i.e., EtOH extracts yielded the greatest total amounts of bufodienolides, and EtOAc precipitation had the lowest amounts of bufodienolides. Toad venom consists of serotonin and several bufodienolides, and the choice of solvent to extract chemical the constituents is important as a way to enrich the purported active components for treating different conditions.

  14. Comparative Analysis of the Bufonis Venenum by Using TLC, HPLC, and LC-MS for Different Extraction Methods

    Directory of Open Access Journals (Sweden)

    Lee Hyo-Jae


    Full Text Available Objectives: Toad venom, called Chan-Su, is a traditional Oriental medicine secreted from the auricular and the skin glands of the Bufo bufo gargarizanz Cantor or B. melanosticus Schneider and has been widely used in China, Korea and other parts of Asia for the treatment of pain, heart conditions, and cancer. We examined the concentrations of the main chemical constituents within a commerciallyavailable toad venom product and compared the levels for different extraction methods. Methods: Toad venom was extracted using either cold or hot water, ethanol (EtOH, methanol (MeOH, or ethyl acetate (EtOAc, was fractionated using precipitation or reflux, and was then analyzed using thin layer chromatography (TLC, high-performance liquid chromatography (HTLC, and liquid chroma-tography - mass spectrometry (LC-MS. Individual components were identified by comparisons of the retention times, the ultraviolet spectra, and mass spectras and differences in chemical constituents for different solvents and extraction methods are presented. Results:Components with authentic standards, including serotonin and bufodienolides (cinobufagen, bufalin, cinobufalin, and resibufogenin, were detected. The water extract of toad venom contained the greatest amount of serotonin (75.7 ± 0.1 mg/g, but very small amounts of bufodienolides (3.8 ± 0.0 mg/g. In contrast, the use of MeOH or EtOH extraction solutions resulted in 5-26 times higher concentrations of bufodienolides, with only trace amounts of serotonin. The relative and the absolute concentrations of the component also varied based on the extraction method; i.e., EtOH extracts yielded the greatest total amounts of bufodienolides, and EtOAc precipitation had the lowest amounts of bufodienolides. Conclusions: Toad venom consists of serotonin and several bufodienolides, and the choice of solvent to extract chemical the constituents is important as a way to enrich the purported active components for treating different

  15. Identification of novel biomarkers for sepsis prognosis via urinary proteomic analysis using iTRAQ labeling and 2D-LC-MS/MS.

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    Longxiang Su

    Full Text Available Sepsis is the major cause of death for critically ill patients. Recent progress in proteomics permits a thorough characterization of the mechanisms associated with critical illness. The purpose of this study was to screen potential biomarkers for early prognostic assessment of patients with sepsis.For the discovery stage, 30 sepsis patients with different prognoses were selected. Urinary proteins were identified using isobaric tags for relative and absolute quantitation (iTRAQ coupled with LC-MS/MS. Mass spec instrument analysis were performed with Mascot software and the International Protein Index (IPI; bioinformatic analyses were used by the algorithm of set and the Gene Ontology (GO Database. For the verification stage, the study involved another 54 sepsis-hospitalized patients, with equal numbers of patients in survivor and non-survivor groups based on 28-day survival. Differentially expressed proteins were verified by Western Blot.A total of 232 unique proteins were identified. Proteins that were differentially expressed were further analyzed based on the pathophysiology of sepsis and biomathematics. For sepsis prognosis, five proteins were significantly up-regulated: selenium binding protein-1, heparan sulfate proteoglycan-2, alpha-1-B glycoprotein, haptoglobin, and lipocalin; two proteins were significantly down-regulated: lysosome-associated membrane proteins-1 and dipeptidyl peptidase-4. Based on gene ontology clustering, these proteins were associated with the biological processes of lipid homeostasis, cartilage development, iron ion transport, and certain metabolic processes. Urinary LAMP-1 was down-regulated, consistent with the Western Blot validation.This study provides the proteomic analysis of urine to identify prognostic biomarkers of sepsis. The seven identified proteins provide insight into the mechanism of sepsis. Low urinary LAMP-1 levels may be useful for early prognostic assessment of NCT01493492.

  16. A proteomics method using immunoaffinity fluorogenic derivatization-liquid chromatography/tandem mass spectrometry (FD-LC-MS/MS) to identify a set of interacting proteins. (United States)

    Nakata, Katsunori; Saitoh, Ryoichi; Ishigai, Masaki; Imai, Kazuhiro


    Biological functions in organisms are usually controlled by a set of interacting proteins, and identifying the proteins that interact is useful for understanding the mechanism of the functions. Immunoprecipitation is a method that utilizes the affinity of an antibody to isolate and identify the proteins that have interacted in a biological sample. In this study, the FD-LC-MS/MS method, which involves fluorogenic derivatization followed by separation and quantification by HPLC and finally identification of proteins by HPLC-tandem mass spectrometry, was used to identify proteins in immunoprecipitated samples, using heat shock protein 90 (HSP90) as a model of an interacting protein in HepaRG cells. As a result, HSC70 protein, which was known to form a complex with HSP90, was isolated, together with three different types of HSP90-beta. The results demonstrated that the proposed immunoaffinity-FD-LC-MS/MS method could be useful for simultaneously detecting and identifying the proteins that interact with a certain protein. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Real Time Extraction Kinetics of Electro Membrane Extraction Verified by Comparing Drug Metabolism Profiles Obtained from a Flow-Flow Electro Membrane Extraction-Mass Spectrometry System with LC-MS

    DEFF Research Database (Denmark)

    Fuchs, David; Jensen, Henrik; Pedersen-Bjergaard, Stig


    A simple to construct and operate, "dip-in" electromembrane extraction (EME) probe directly coupled to electrospray ionization-mass spectrometry (ESI-MS) for rapid extraction and real time analysis of various analytes was developed. The setup demonstrated that EME-MS can be used as a viable...... alternative to conventional protein precipitation followed by liquid chromatography-mass spectrometry (LC-MS) for studying drug metabolism. Comparison of EME-MS with LC-MS for drug metabolism analysis demonstrated for the first time that real time extraction of analytes by EME is possible. Metabolism kinetics...... were investigated for three different drugs: amitriptyline, promethazine, and methadone. By comparing the EME-MS extraction profiles of the drug substances and formed drug metabolites with the metabolism profiles obtained by conventional protein precipitation followed by LC-MS good correlation...

  18. LC-MS based Metabolomics

    DEFF Research Database (Denmark)

    Magdenoska, Olivera

    metabolites with liquid chromatography mass spectrometry (LC-MS) as the most commonly used. The primary goal of this Ph.D. study was to develop an LC-MS method together with sample preparation for analysis of intracellular metabolites such as nucleotides, sugar phosphates, organic acids, coenzymes etc...... during this Ph.D. study were successfully applied for targeted and multitargeted analysis of different classes of intracellular metabolites such as nucleotides, sugar phosphates, coeznymes and organic acids. In addition sample preparation methods were established for different microorganisms capable...... recovery of the metabolites after the extraction. Quenching and extraction procedures for bacteria, yeast, mammalian cells and filamentous fungi were tested. Cold MeOH as a quenching method combined with boiling EtOH or MeOH/chloroform as extraction method showed to work well for Saccharomyces cerevisiae...

  19. An atmospheric pressure ionization source using a high voltage target compared to electrospray ionization for the LC/MS analysis of pharmaceutical compounds. (United States)

    Lubin, Arnaud; De Vries, Ronald; Cabooter, Deirdre; Augustijns, Patrick; Cuyckens, Filip


    The type and design of an ionization source can have a significant influence on the performances of a bioanalytical method. It is, therefore, of high interest to evaluate the performances of newly introduced sources to highlight their benefits and limitations in comparison to other well established sources. In this paper, liquid chromatography - mass spectrometry (LC/MS) performances of a new atmospheric pressure ionization (API) source, commercialized as UniSpray, is evaluated. The dynamic range of 24 pharmaceutical and biological compounds is compared between the new API source and electrospray ionization (ESI) for 3 different mobile phase conditions. Matrix effects are also compared with ESI on a refined selection of 19 pharmaceutical and biological compounds in 4 matrices commonly encountered in bioanalysis. A slightly better dynamic range towards lower concentrations was often observed with the new API source. Matrix effects were quite similar between the two sources with a small, but statistically significant, lower percentage of matrix effects observed for the new API source in plasma and bile in the positive ion mode, and bile in negative ion mode for ESI. Finally, the sensitivity of late eluting compounds could be improved on the new API source by post-column addition of water. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Identification of recombinant human EPO variants in greyhound plasma and urine by ELISA, LC-MS/MS and western blotting: a comparative study. (United States)

    Timms, Mark; Steel, Rohan; Vine, John


    The recombinant human erythropoietins epoetin alfa (Eprex®), darbepoetin (Aranesp®) and methoxy polyethylene glycol-epoetin beta (Mircera®) were administered to greyhounds for 7, 10 and 14 days respectively. Blood and urine samples were collected and analysed for erythropoietin by ELISA, LC-MS/MS and western blotting. Limits of confirmation in plasma for western blotting and LC-MS/MS methods ranged from a low of 2.5mIU/mL, and closely matched the sensitivity of ELISA screening. Copyright © 2015 John Wiley & Sons, Ltd.

  1. AMDORAP: Non-targeted metabolic profiling based on high-resolution LC-MS

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    Ogasawara Naotake


    Full Text Available Abstract Background Liquid chromatography-mass spectrometry (LC-MS utilizing the high-resolution power of an orbitrap is an important analytical technique for both metabolomics and proteomics. Most important feature of the orbitrap is excellent mass accuracy. Thus, it is necessary to convert raw data to accurate and reliable m/z values for metabolic fingerprinting by high-resolution LC-MS. Results In the present study, we developed a novel, easy-to-use and straightforward m/z detection method, AMDORAP. For assessing the performance, we used real biological samples, Bacillus subtilis strains 168 and MGB874, in the positive mode by LC-orbitrap. For 14 identified compounds by measuring the authentic compounds, we compared obtained m/z values with other LC-MS processing tools. The errors by AMDORAP were distributed within ±3 ppm and showed the best performance in m/z value accuracy. Conclusions Our method can detect m/z values of biological samples much more accurately than other LC-MS analysis tools. AMDORAP allows us to address the relationships between biological effects and cellular metabolites based on accurate m/z values. Obtaining the accurate m/z values from raw data should be indispensable as a starting point for comparative LC-orbitrap analysis. AMDORAP is freely available under an open-source license at

  2. Real Time Extraction Kinetics of Electro Membrane Extraction Verified by Comparing Drug Metabolism Profiles Obtained from a Flow-Flow Electro Membrane Extraction-Mass Spectrometry System with LC-MS. (United States)

    Fuchs, David; Jensen, Henrik; Pedersen-Bjergaard, Stig; Gabel-Jensen, Charlotte; Hansen, Steen Honoré; Petersen, Nickolaj Jacob


    A simple to construct and operate, "dip-in" electromembrane extraction (EME) probe directly coupled to electrospray ionization-mass spectrometry (ESI-MS) for rapid extraction and real time analysis of various analytes was developed. The setup demonstrated that EME-MS can be used as a viable alternative to conventional protein precipitation followed by liquid chromatography-mass spectrometry (LC-MS) for studying drug metabolism. Comparison of EME-MS with LC-MS for drug metabolism analysis demonstrated for the first time that real time extraction of analytes by EME is possible. Metabolism kinetics were investigated for three different drugs: amitriptyline, promethazine, and methadone. By comparing the EME-MS extraction profiles of the drug substances and formed drug metabolites with the metabolism profiles obtained by conventional protein precipitation followed by LC-MS good correlation was obtained with only very limited time delay in the extraction. The results indicate that, by tuning the electromembrane properties, for example, by optimizing the extraction voltage, extremely fast extraction kinetics can be obtained. A metabolic profile could be generated while the drug was metabolized offering a significant time saving as compared to conventional LC-MS where laborious protein precipitation or other sample pretreatments are required before analysis. This makes the developed EME-MS setup a highly promising sample preparation method for various kinds of applications where fast and real-time analysis of analytes is of interest.

  3. Experimental design and data-analysis in label-free quantitative LC/MS proteomics: A tutorial with MSqRob. (United States)

    Goeminne, Ludger J E; Gevaert, Kris; Clement, Lieven


    Label-free shotgun proteomics is routinely used to assess proteomes. However, extracting relevant information from the massive amounts of generated data remains difficult. This tutorial provides a strong foundation on analysis of quantitative proteomics data. We provide key statistical concepts that help researchers to design proteomics experiments and we showcase how to analyze quantitative proteomics data using our recent free and open-source R package MSqRob, which was developed to implement the peptide-level robust ridge regression method for relative protein quantification described by Goeminne et al. MSqRob can handle virtually any experimental proteomics design and outputs proteins ordered by statistical significance. Moreover, its graphical user interface and interactive diagnostic plots provide easy inspection and also detection of anomalies in the data and flaws in the data analysis, allowing deeper assessment of the validity of results and a critical review of the experimental design. Our tutorial discusses interactive preprocessing, data analysis and visualization of label-free MS-based quantitative proteomics experiments with simple and more complex designs. We provide well-documented scripts to run analyses in bash mode on GitHub, enabling the integration of MSqRob in automated pipelines on cluster environments ( The concepts outlined in this tutorial aid in designing better experiments and analyzing the resulting data more appropriately. The two case studies using the MSqRob graphical user interface will contribute to a wider adaptation of advanced peptide-based models, resulting in higher quality data analysis workflows and more reproducible results in the proteomics community. We also provide well-documented scripts for experienced users that aim at automating MSqRob on cluster environments. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Allium cepa L. Response to Sodium Selenite (Se(IV)) Studied in Plant Roots by a LC-MS-Based Proteomic Approach. (United States)

    Karasinski, Jakub; Wrobel, Kazimierz; Corrales Escobosa, Alma Rosa; Konopka, Anna; Bulska, Ewa; Wrobel, Katarzyna


    Liquid chromatography-high-resolution mass spectrometry was used for the first time to investigate the impact of Se(IV) (10 mgSe L -1 as sodium selenite) on Allium cepa L. root proteome. Using MaxQuant platform, more than 600 proteins were found; 42 were identified based on at least 2 razor + unique peptides, score > 25, and were found to be differentially expressed in the exposed versus control roots with t-test difference > ±0.70 (p roots. Different abundances of proteins involved in transcriptional regulation, protein folding/assembly, cell cycle, energy/carbohydrate metabolism, stress response, and antioxidant defense were found in the exposed vs nonexposed roots. New evidence was obtained on the alteration of sulfur metabolism due to S-Se competition in A. cepa L. which, together with the original analytical approach, is the main scientific contribution of this study. Specifically, proteins participating in assimilation and transformation of both elements were affected; formation of volatile Se compounds seemed to be favored. Changes observed in methionine cycle suggested that Se(IV) stress might repress methylation capability in A. cepa L., potentially limiting accumulation of Se in the form of nonprotein methylated species and affecting adversely transmethylation-dependent signaling pathways.

  5. Processing methods for differential analysis of LC/MS profile data

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    Orešič Matej


    Full Text Available Abstract Background Liquid chromatography coupled to mass spectrometry (LC/MS has been widely used in proteomics and metabolomics research. In this context, the technology has been increasingly used for differential profiling, i.e. broad screening of biomolecular components across multiple samples in order to elucidate the observed phenotypes and discover biomarkers. One of the major challenges in this domain remains development of better solutions for processing of LC/MS data. Results We present a software package MZmine that enables differential LC/MS analysis of metabolomics data. This software is a toolbox containing methods for all data processing stages preceding differential analysis: spectral filtering, peak detection, alignment and normalization. Specifically, we developed and implemented a new recursive peak search algorithm and a secondary peak picking method for improving already aligned results, as well as a normalization tool that uses multiple internal standards. Visualization tools enable comparative viewing of data across multiple samples. Peak lists can be exported into other data analysis programs. The toolbox has already been utilized in a wide range of applications. We demonstrate its utility on an example of metabolic profiling of Catharanthus roseus cell cultures. Conclusion The software is freely available under the GNU General Public License and it can be obtained from the project web page at:

  6. Quantitative proteomic analysis of Pseudomonas pseudoalcaligenes CECT5344 in response to industrial cyanide-containing wastewaters using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS.

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    María Isabel Ibáñez

    Full Text Available Biological treatments to degrade cyanide are a powerful technology for cyanide removal from industrial wastewaters. It has been previously demonstrated that the alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to use free cyanide and several metal-cyanide complexes as the sole nitrogen source. In this work, the strain CECT5344 has been used for detoxification of the different chemical forms of cyanide that are present in alkaline wastewaters from the jewelry industry. This liquid residue also contains large concentrations of metals like iron, copper and zinc, making this wastewater even more toxic. To elucidate the molecular mechanisms involved in the bioremediation process, a quantitative proteomic analysis by LC-MS/MS has been carried out in P. pseudoalcaligenes CECT5344 cells grown with the jewelry residue as sole nitrogen source. Different proteins related to cyanide and cyanate assimilation, as well as other proteins involved in transport and resistance to metals were induced by the cyanide-containing jewelry residue. GntR-like regulatory proteins were also induced by this industrial residue and mutational analysis revealed that GntR-like regulatory proteins may play a role in the regulation of cyanide assimilation in P. pseudoalcaligenes CECT5344. The strain CECT5344 has been used in a batch reactor to remove at pH 9 the different forms of cyanide present in industrial wastewaters from the jewelry industry (0.3 g/L, ca. 12 mM total cyanide, including both free cyanide and metal-cyanide complexes. This is the first report describing the biological removal at alkaline pH of such as elevated concentration of cyanide present in a heterogeneous mixture from an industrial source.

  7. Differential Proteomic Analysis of Syncytiotrophoblast Extracellular Vesicles from Early-Onset Severe Preeclampsia, using 8-Plex iTRAQ Labeling Coupled with 2D Nano LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Hongmei Li


    Full Text Available Aims: Previous studies have revealed that the increased shedding of syncytiotrophoblast extracellular vesicles (STBM may lead to preeclampsia (PE. We aimed to identify the proteins carried by STBM and their potential pathological roles in early-onset severe PE. Methods: In this study, we performed a differential proteomic analysis of STBM from early-onset severe PE patients, using iTRAQ isobaric tags and 2D nano LC-MS/MS. STBM were generated by the in vitro explant culture method, and then verified by electron microscopy and western blot analysis. Results: A total of 18 533 unique peptides and 3 317 proteins were identified, 3 292 proteins were quantified. We identified 194 differentially expressed proteins in STBM from early-onset severe PE patients, 122 proteins were up-regulated and 72 proteins were down-regulated. Further bioinformatics analysis revealed that mitochondrion, transmembrane transport and transmembrane transporter activity were the most abundant categories in gene ontology (GO annotation. Glycolysis/ gluconeogenesis, citrate cycle, fatty acid elongation, steroid hormone biosynthesis and oxidative phosphorylation were the five significantly represented pathways. Four differentially expressed proteins (siglec-6, calnexin, CD63 and S100-A8 related to inflammation, coagulation or immunoregulation were independently verified using western blot. Conclusions: The identification of key proteins carried by STBM may serve not only as a basis for better understanding and further exploring the etiology and pathogenesis of PE, but also as potential biomarkers and in providing targets for future therapy in PE, especially in early-onset severe PE(sPE.

  8. A comparative tissue-specific metabolite analysis and determination of protodioscin content in Asparagus species used in traditional Chinese medicine and Ayurveda by use of laser microdissection, UHPLC-QTOF/MS and LC-MS/MS. (United States)

    Jaiswal, Yogini; Liang, Zhitao; Ho, Alan; Chen, Hubiao; Zhao, Zhongzhen


    Asparagus is esteemed in Traditional Chinese Medicine and Ayurveda, and it is commercially one of the most important drugs in the global herbal market. Comparative metabolite profiling of different species would help in determining the similarities and ascertain their validity for being used as substitutes for each other. Laser microdissection (LMD) facilitates identification of metabolites in specific tissues, and thus it can aid in exploration of metabolic pathways in target tissues. To compare tissue-specific metabolites and protodioscin content of Asparagus cochinchinensis (Lour.) Merr. and Asparagus racemosus Willd. used in China and India. Metabolite analysis of laser-dissected tissues was carried out using UHPLC-QTOF/MS and LC-MS/MS. The protodioscin contents were determined and the method was validated as per the International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines. Metabolite analysis reveals that the velamen tissue, among other tissues such as cortex, vascular bundles and pith, contained maximum components, specifically those belonging to the steroidal saponin class. Although the metabolite profiles were similar, the content of protodioscin was found to be higher in Chinese than Indian species. The study provided a suitable methodology for metabolite profiling and protodioscin content determination of Asparagus by use of LMD, UHPLC-QTOF/MS and LC-MS/MS. The similarities in metabolite profiles indicate that Asparagus species from India and China can serve as substitute for each other in various therapeutic and pharmaceutical applications. Copyright © 2014 John Wiley & Sons, Ltd.

  9. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products. (United States)

    Hegele, Jörg; Buetler, Timo; Delatour, Thierry


    Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N(epsilon)-fructoselysine (FL), N(epsilon)-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34+/-3.81 nmol CML per micromol of free Lys (Lys(free)) and 81.5+/-87.8 nmol Pyr micromol(-1) Lys(free)(-1) vs. 3.72+/-1.29 nmol FL micromol(-1) Lys(free)(-1). In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47+/-0.08 nmol FL micromol(-1) of protein-bound Lys (Lys(p-b)), 0.04+/-0.03 nmol CML micromol(-1) Lys(p-b)(-1) and 0.06+/-0.02 nmol Pyr micromol(-1)Lys(p-b)(-1). It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

  10. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

    International Nuclear Information System (INIS)

    Hegele, Joerg; Buetler, Timo; Delatour, Thierry


    Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N ε -fructoselysine (FL), N ε -carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 ± 3.81 nmol CML per μmol of free Lys (Lys free ) and 81.5 ± 87.8 nmol Pyr μmol -1 Lys free -1 vs. 3.72 ± 1.29 nmol FL μmol -1 Lys free -1 . In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 ± 0.08 nmol FL μmol -1 of protein-bound Lys (Lys p-b ), 0.04 ± 0.03 nmol CML μmol -1 Lys p-b -1 and 0.06 ± 0.02 nmol Pyr μmol -1 Lys p-b -1 . It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products

  11. Comparative LC-MS/MS profiling of free and protein-bound early and advanced glycation-induced lysine modifications in dairy products

    Energy Technology Data Exchange (ETDEWEB)

    Hegele, Joerg [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)], E-mail:; Buetler, Timo; Delatour, Thierry [Nestle Research Centre, Nestec Ltd., Vers-chez-les-Blanc, CH-1000 Lausanne 26 (Switzerland)


    Free and protein-bound forms of early and advanced glycation-induced lysine (Lys) modifications were quantified in dairy products by LC-MS/MS using a stable isotope dilution assay. The glycation profiles for N{sup {epsilon}}-fructoselysine (FL), N{sup {epsilon}}-carboxymethyllysine (CML) and pyrraline (Pyr) were monitored in raw and processed cow milk to investigate whether free glycation products could serve as fast and simple markers to assess the extent of protein glycation in dairy products. In all milk samples, the fraction of free glycation adducts was predominantly composed of advanced modifications, e.g. 8.34 {+-} 3.81 nmol CML per {mu}mol of free Lys (Lys{sub free}) and 81.5 {+-} 87.8 nmol Pyr {mu}mol{sup -1} Lys{sub free}{sup -1} vs. 3.72 {+-} 1.29 nmol FL {mu}mol{sup -1} Lys{sub free}{sup -1}. In contrast, the protein-bound early glycation product FL considerably outweighed the content of CML and Pyr in milk proteins of raw and processed cow milk, whereas severely heat treated milk products, e.g. condensed milk, contained a higher amount of protein-bound advanced glycation adducts. Typical values recorded for milk samples processed under mild conditions were 0.47 {+-} 0.08 nmol FL {mu}mol{sup -1} of protein-bound Lys (Lys{sub p-b}), 0.04 {+-} 0.03 nmol CML {mu}mol{sup -1} Lys{sub p-b}{sup -1} and 0.06 {+-} 0.02 nmol Pyr {mu}mol{sup -1} Lys{sub p-b}{sup -1}. It was particularly noticeable, however, that mild heat treatment of raw milk, i.e. pasteurization and UHT treatment, did not significantly increase the amount of both free and protein-bound Lys modifications. In conclusion, the profiles of free and protein-bound glycation-induced Lys modifications were found to be different and a screening of free glycation adducts does, therefore, not allow for a conclusion about the protein glycation status of dairy products.

  12. Direct coupling of a flow-flow electromembrane extraction probe to LC-MS

    DEFF Research Database (Denmark)

    Fuchs, David; Gabel-Jensen, Charlotte; Jensen, Henrik


    of methadone by rat liver microsomes. As the metabolic reaction proceeded, methadone and its metabolites were extracted and analyzed in parallel by LC-MS using either isocratic or gradient elution. Compared to a conventional in-vitro metabolism analysis based on protein precipitation followed by LC-MS analysis...

  13. Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes. (United States)

    Jin, Ya; Yuan, Qi; Zhang, Jun; Manabe, Takashi; Tan, Wen


    Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel-cutting, and quantitative LC-MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an "overlap score," (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 "overlap factors," (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Comparative Proteomic Analysis of Hymenolepis diminuta Cysticercoid and Adult Stages

    Directory of Open Access Journals (Sweden)

    Anna Sulima


    Full Text Available Cestodiases are common parasitic diseases of animals and humans. As cestodes have complex lifecycles, hexacanth larvae, metacestodes (including cysticercoids, and adults produce proteins allowing them to establish invasion and to survive in the hostile environment of the host. Hymenolepis diminuta is the most commonly used model cestode in experimental parasitology. The aims of the present study were to perform a comparative proteomic analysis of two consecutive developmental stages of H. diminuta (cysticercoid and adult and to distinguish proteins which might be characteristic for each of the stages from those shared by both stages. Somatic proteins of H. diminuta were isolated from 6-week-old cysticercoids and adult tapeworms. Cysticercoids were obtained from experimentally infected beetles, Tenebrio molitor, whereas adult worms were collected from experimentally infected rats. Proteins were separated by GeLC-MS/MS (one dimensional gel electrophoresis coupled with liquid chromatography and tandem mass spectrometry. Additionally protein samples were digested in-liquid and identified by LC-MS/MS. The identified proteins were classified according to molecular function, cellular components and biological processes. Our study showed a number of differences and similarities in the protein profiles of cysticercoids and adults; 233 cysticercoid and 182 adult proteins were identified. From these proteins, 131 were present only in the cysticercoid and 80 only in the adult stage samples. Both developmental stages shared 102 proteins; among which six represented immunomodulators and one is a potential drug target. In-liquid digestion and LC-MS/MS complemented and confirmed some of the GeLC-MS/MS identifications. Possible roles and functions of proteins identified with both proteomic approaches are discussed.

  15. A new reliable, transposable and cost-effective assay for absolute quantification of total plasmatic bevacizumab by LC-MS/MS in human plasma comparing two internal standard calibration approaches. (United States)

    Legeron, Rachel; Xuereb, Fabien; Chaignepain, Stephane; Gadeau, Alain-Pierre; Claverol, Stephane; Dupuy, Jean-William; Djabarouti, Sarah; Couffinhal, Thierry; Schmitter, Jean-Marie; Breilh, Dominique


    The quantification of monoclonal antibodies (mAbs) such as bevacizumab, a recombinant humanized immunoglobulin G1 (hIgG1), in biological fluids, is an essential prerequisite to any pharmacokinetic preclinical and clinical study. To date, reference techniques used to quantify mAbs rely on enzyme-linked immunosorbent assay (ELISA) lacking specificity. Furthermore, the commercially available ELISA kit to quantify bevacizumab in human plasma only assesses the free fraction of the drug. However, the conditions of storage and analysis of plasma samples could alter the physiological equilibrium between the free, bound and partially bound forms of bevacizumab and this could result in over- or underestimation of drug concentration. We developed a new assay for absolute quantification of total fraction of bevacizumab by liquid chromatography tandem mass spectrometry (LC-MS/MS) basing identification and quantification of bevacizumab on two specific peptides. In this report we compare our assay with two internal standard (IS) calibration approaches: one using a different human mAb (Trastuzumab) and the other using a stable isotope labeled specific peptide. After enrichment by affinity chromatography on protein A and concentration by ultrafiltration, human plasma samples were proteolyzed by trypsin. Linearity was established from 12.5 to 500μg/mL with an interday accuracy ranging from 101.7 to 110.6% and precision from 7.0% to 9.9%. This study demonstrates the importance of the choice of the IS in quantifying bevacizumab in human plasma and highlights the difficulty of reaching a reliable proteolysis with a sufficient recovery. We developed a reliable and cost-effective LC-MS/MS method to quantify total plasmatic fraction of bevacizumab in human plasma. Through our development we proposed a generic methodology easily transposable to quantify all IgG1 subclass very useful for clinical pharmacokinetics studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hui; Shi, Tujin; Qian, Wei-Jun; Liu, Tao; Kagan, Jacob; Srivastava, Sudhir; Smith, Richard D.; Rodland, Karin D.; Camp, David G.


    Mass spectrometry-based proteomics has become an indispensable tool in biomedical research with broad applications ranging from fundamental biology, systems biology, and biomarker discovery. Recent advances in LC-MS have made it become a major technology in clinical applications, especially in cancer biomarker discovery and verification. To overcome the challenges associated with the analysis of clinical samples, such as extremely wide dynamic range of protein concentrations in biofluids and the need to perform high throughput and accurate quantification, significant efforts have been devoted to improve the overall performance of LC-MS bases clinical proteomics. In this review, we summarize the recent advances in LC-MS in the aspect of cancer biomarker discovery and quantification, and discuss its potentials, limitations, and future perspectives.

  17. Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC-MS/MS. 1. Preparation of more than 4000 native protein maps. (United States)

    Jin, Ya; Zhang, Jun; Yuan, Qi; Manabe, Takashi; Tan, Wen


    Soluble proteins of human bronchial smooth muscle cells (HBSMC) were separated by nondenaturing micro 2DE and a 30 mm × 40 mm area of the CBB-stained slab gel (1.0 mm thick) was cut into 1.1 mm × 1.1 mm squares, then the proteins in the 972 gel pieces (squares) were applied to quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data [1]. Totally 4323 proteins were identified in successfully analyzed 967 squares and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The quantity of the proteins distributed from 3.6% to 1 × 10(-5) % of the total protein quantity in the grid area. Each protein map was characterized by several features, including the position of quantity peak square, number of detected squares, and degree of concentration (focused or dispersed). About 4% of the proteins were detected in 100 or more squares, suggesting that they might be ubiquitous and interacting with other proteins. In contrast, many proteins showed more concentrated quantity distribution and the quantity peak positions of 565 proteins with a defined degree of concentration were summarized into a quantity peak map. These results for the first time visualized the distribution patterns of cellular proteins on a nondenaturing 2D gel. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Analysis of pesticide residues in strawberries and soils by GC-MS/MS, LC-MS/MS and two-dimensional GC-time-of-flight MS comparing organic and integrated pest management farming. (United States)

    Fernandes, Virgínia C; Lehotay, Steven J; Geis-Asteggiante, Lucía; Kwon, Hyeyoung; Mol, Hans G J; van der Kamp, Henk; Mateus, Nuno; Domingues, Valentina F; Delerue-Matos, Cristina


    This study analysed 22 strawberry and soil samples after their collection over the course of 2 years to compare the residue profiles from organic farming with integrated pest management practices in Portugal. For sample preparation, we used the citrate-buffered version of the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method. We applied three different methods for analysis: (1) 27 pesticides were targeted using LC-MS/MS; (2) 143 were targeted using low pressure GC-tandem mass spectrometry (LP-GC-MS/MS); and (3) more than 600 pesticides were screened in a targeted and untargeted approach using comprehensive, two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC-TOF-MS). Comparison was made of the analyses using the different methods for the shared samples. The results were similar, thereby providing satisfactory confirmation of both similarly positive and negative findings. No pesticides were found in the organic-farmed samples. In samples from integrated pest management practices, nine pesticides were determined and confirmed to be present, ranging from 2 µg kg(-1) for fluazifop-p-butyl to 50 µg kg(-1) for fenpropathrin. Concentrations of residues in strawberries were less than European maximum residue limits.

  19. Combining Search Engines for Comparative Proteomics (United States)

    Tabb, David


    Many proteomics laboratories have found spectral counting to be an ideal way to recognize biomarkers that differentiate cohorts of samples. This approach assumes that proteins that differ in quantity between samples will generate different numbers of identifiable tandem mass spectra. Increasingly, researchers are employing multiple search engines to maximize the identifications generated from data collections. This talk evaluates four strategies to combine information from multiple search engines in comparative proteomics. The “Count Sum” model pools the spectra across search engines. The “Vote Counting” model combines the judgments from each search engine by protein. Two other models employ parametric and non-parametric analyses of protein-specific p-values from different search engines. We evaluated the four strategies in two different data sets. The ABRF iPRG 2009 study generated five LC-MS/MS analyses of “red” E. coli and five analyses of “yellow” E. coli. NCI CPTAC Study 6 generated five concentrations of Sigma UPS1 spiked into a yeast background. All data were identified with X!Tandem, Sequest, MyriMatch, and TagRecon. For both sample types, “Vote Counting” appeared to manage the diverse identification sets most effectively, yielding heightened discrimination as more search engines were added.

  20. Development and validation of LC-MS/MS assay for the determination of the prodrug Midodrine and its active metabolite Desglymidodrine in plasma of ascitic patients: Application to individualized therapy and comparative pharmacokinetics. (United States)

    Ali, Ahmed A; Al-Ghobashy, Medhat A; Farid, Samar F; Kassem, Mohamed A


    Midodrine (MD) is a prodrug that is converted after oral administration to Desglymidodrine (DMD). In this study, an LC-MS/MS assay was developed and validated for investigation of the pharmacokinetics of MD and DMD in non azotemic patients with liver cirrhosis and tense ascites. Results were compared to those noted with healthy volunteers following the adminstration of a single oral dose of MD. Sample preparation was performed by liquid-liquid extraction using t-butyl methyl ether. HPLC separation was carried out using RP C18 column (4.6mm×50mm, 5μm). Isocratic elution was performed using methanol:0.2% formic acid (70:30, v/v) as the mobile phase, at a flow rate of 0.7mL/min. Tandem mass spectrometric detection was employed at positive electrospray ionization in MRM mode for the determination of MD and DMD. Analysis was carried out within 1.0min over a concentration range of 0.50-40.00ng/mL for the prodrug and its active metabolite. The assay was validated according to FDA guidelines for bioanalytical method validation and satisfactory results were obtained. The applicability of the assay for the determination of the pharmacokinetic parameters of MD and DMD and personalized therapy was demonstrated in healthy volunteers and ascitic patients. Results revealed significant differences in pharmacokinetic parameters among the studied groups. Such differences were explained on the basis of the medical condition and co-adminstered medications exerting possible drug-drug interaction. Results confirmed the need for implementation of reliable analysis tools for therapeutic dose adjustment. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Gluten Detection and Speciation by Liquid Chromatography Mass Spectrometry (LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Stephen Lock


    Full Text Available Liquid chromatography tandem mass spectrometry (LC-MS/MS has been used historically in proteomics research for over 20 years. However, until recently LC-MS/MS has only been routinely used in food testing for small molecule contaminant detection, for example pesticide and veterinary residue detection, and not as a replacement of microbiological food testing methods, specifically allergen analysis. Over the last couple of years, articles have started to be published which describe the detection of allergens by LC-MS/MS. In this article we will describe how LC-MS/MS can be applied in the area of gluten detection and how it can be used to specifically differentiate the species of gluten used in food, where specific markers for each variety of gluten can be simultaneously acquired and detected at the same time. The article will discuss the effect of variety on the peptide response observed from different wheat grain varieties and will describe the sample preparation protocol which is essential for generating the peptide markers used for speciation.

  2. LC-MS/MS determination and comparative pharmacokinetics of strychnine, brucine and their metabolites in rat plasma after intragastric administration of each monomer and the total alkaloids from Semen Strychni. (United States)

    Lin, Aihua; Su, Xiaochun; She, Dan; Qiu, Kuncheng; He, Qianmei; Liu, Yiming


    A rapid, specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous determination of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma. Plasma samples were pretreated via simple protein precipitation with methanol and ephedrine hydrochloride was used as internal standard. Chromatographic separation was carried out on an ZORBAX Eclipse XDB-C18 column (2.1×150mm, 3.5μm) by gradient elution with methanol and 10mM ammonium acetate (adjusted to pH 4.0 with formic acid). The quantification of the analytes was performed by mass spectrometry with TurboIonSpray ionization (ESI) inlet in the positive ion multiple reaction monitoring (MRM) mode. The results showed that the calibration curve was linear in the concentration range of 0.510∼306.3ngmL(-1) for strychnine, brucine and 0.102∼306.0ngmL(-1) for strychnine N-oxide and brucine N-oxide, respectively. The intra- and inter-day precisions were less than 14.9%, and the accuracy ranged from 89.4 to 113% at three QC levels for the 4 analytes. The validated method was successfully applied to the pharmacokinetic study of strychnine, brucine, strychnine N-oxide and brucine N-oxide in rat plasma after oral administration of each monomer and the total alkaloids from Semen Strychni. After single oral administration of the total alkaloids from Semen Strychni at 4 dose levels, Cmax, AUC0-t of strychnine and brucine increased and were proportional to the oral doses. In comparative pharmacokinetics studies, no significant difference was found between each monomer and the total strychnos alkaloids on the pharmacokinetic parameters such as Cmax and AUC. Mean Cmax and AUC of strychnine and brucine were slight increased in the monomer groups in comparison to the total strychnos alkaloids groups, which suggested that some other alkaloids in the Semen Strychni may decrease the absorption of strychnine and brucine in body. Copyright

  3. A Proof of Concept to Bridge the Gap between Mass Spectrometry Imaging, Protein Identification and Relative Quantitation: MSI~LC-MS/MS-LF

    Directory of Open Access Journals (Sweden)

    Laëtitia Théron


    Full Text Available Mass spectrometry imaging (MSI is a powerful tool to visualize the spatial distribution of molecules on a tissue section. The main limitation of MALDI-MSI of proteins is the lack of direct identification. Therefore, this study focuses on a MSI~LC-MS/MS-LF workflow to link the results from MALDI-MSI with potential peak identification and label-free quantitation, using only one tissue section. At first, we studied the impact of matrix deposition and laser ablation on protein extraction from the tissue section. Then, we did a back-correlation of the m/z of the proteins detected by MALDI-MSI to those identified by label-free quantitation. This allowed us to compare the label-free quantitation of proteins obtained in LC-MS/MS with the peak intensities observed in MALDI-MSI. We managed to link identification to nine peaks observed by MALDI-MSI. The results showed that the MSI~LC-MS/MS-LF workflow (i allowed us to study a representative muscle proteome compared to a classical bottom-up workflow; and (ii was sparsely impacted by matrix deposition and laser ablation. This workflow, performed as a proof-of-concept, suggests that a single tissue section can be used to perform MALDI-MSI and protein extraction, identification, and relative quantitation.

  4. Identification of adulterants in a Chinese herbal medicine by LC-HRMS and LC-MS-SPE/NMR and comparative in vivo study with standards in a hypertensive rat model

    DEFF Research Database (Denmark)

    Kesting, Julie Regitze; Huang, JingQi; Sørensen, Dan


    Based on anecdotal evidence of anti-hypertensive effect of Gold Nine Soft Capsules, an in vivo study of this complex Chinese "herbal-based" medicine was initiated. Dosage of the content of Gold Nine capsules in spontaneous hypertensive rats showed a remarkably good effect. This led to further...... of a combination of commercially purchased standards was shown to be equivalent to that of the capsule content. Adulteration of herbal remedies and dietary supplements with synthetic drugs is an increasing problem that may lead to serious adverse effects. LC-MS-SPE/NMR as a method for the rapid identification...

  5. Novel LC-MS/MS method for plasma vancomycin: comparison with immunoassays and clinical impact. (United States)

    Oyaert, Matthijs; Peersman, Nele; Kieffer, Davy; Deiteren, Kathleen; Smits, Anne; Allegaert, Karel; Spriet, Isabel; Van Eldere, Johan; Verhaegen, Jan; Vermeersch, Pieter; Pauwels, Steven


    Accurate quantification of vancomycin in plasma is important for adequate dose-adjustment. As literature suggests between-method differences, our first objective was to develop a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for total vancomycin in human plasma and to compare frequently used immunoassays with this method. Secondly, we investigated the clinical impact of between-method quantification differences. For LC-MS/MS, lithium heparin plasma was extracted by adding a precipitation reagent containing the internal standard (vancomycin-des-leucine). Analysis was performed on an Acquity TQD mass spectrometer equipped with an Acquity UPLC 2795 separations module. Our method was analytically validated and compared with four frequently used immunoassays from four different manufacturers. Vancomycin concentrations were clinically classified as toxic, therapeutic and sub-therapeutic. Clinical discordance was calculated using LC-MS/MS as a reference. A novel LC-MS/MS method using protein precipitation as sole pretreatment and an analysis time of 5.0 min was developed. The assay had a total imprecision of 2.6-8.5%, a limit of quantification of 0.3 mg/L and an accuracy ranging from 101.4 to 111.2%. Using LC-MS/MS as reference, three immunoassays showed a mean proportional difference within 10% and one showed a substantial mean proportional difference of >20%. Clinical discordant interpretation of the obtained concentrations ranged from 6.1 to 22.2%. We developed a novel LC-MS/MS method for rapid analysis of total vancomycin concentrations in human plasma. Correlation of the method with immunoassays showed a mean proportional difference >20% for one of the assays, causing discordant clinical interpretation in more than 1 out of 5 samples. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Comparative proteomic analysis of longissimus dorsi muscle in immuno- and surgically castrated male pigs. (United States)

    Shi, Xuebin; Li, Chunbao; Cao, Miaodan; Xu, Xinglian; Zhou, Guanghong; Xiong, Youling L


    We compared proteomic profiles of male pig muscles after active immunization against gonadotropin releasing hormone (GnRH) and surgical castration. Longissimus dorsi samples were collected from immunocastration (IC) and surgical castration (SC) groups (n=15 each). Muscle proteins were extracted and then identified by data-independent label-free nano LC-MS/MS. A total of 610 proteins were identified, 50 of which were differentially expressed (Pimmunity were abundant in IC group. Several heat shock proteins (HSPs) and laminins were abundant in SC group. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. The 27th Montreux Symposium on LC-MS. (United States)

    van der Greef, Jan


    The recent Montreux Symposium on LC-MS held in 2010 provided an insight into the key areas of present achievements and future developments in LC-MS. This domain is rapidly changing from a technology- to an application-driven enterprise. New demands for advanced options are coming from (medical) biology, biotechnology, pharmaceutical and nutrition research. In particular, new diagnostic options based on -omics technologies, miniaturization and advanced data handling are rapidly being developed in relation to questions coming from personalized medicine, claim support in nutritional research, biotechnology production support and the human microbiome.

  8. LC-MS characterization of constituents of mesquite flour (United States)

    Using an LC-MS method in conjunction with two complementary types of chromatographic retention modes—namely reversed phase and aqueous normal phase (ANP)—various compounds present in mesquite flour extracts were identified. Because of the diverse types of chemical constituents found in such natural ...

  9. LC-MS at core of university-industry link

    DEFF Research Database (Denmark)

    Linding, Rune


    LC-MS at core of university-industry link Thermo Fisher Scientific (TFS) and the Department of Systems Biology at the Technical University of Denmark, (DTU), have formed a collaboration to pursue breakthroughs in the understanding of how cellular protein networks drive important diseases by explo...

  10. Aflatoxins contamination in Pakistani brown rice: a comparison of TLC, HPLC, LC-MS/MS and ELISA techniques. (United States)

    Iqbal, Javed; Asghar, Muhammad Asif; Ahmed, Aftab; Khan, Mobeen Ahmed; Jamil, Khalid


    Advancement in the field of analytical food-chemistry has explored various experimental techniques for aflatoxins (AFs) quantification. The present study was aimed to compare four different techniques; thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) for the analysis of aflatoxin B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) in brown rice (n = 120) being collected from Karachi, Pakistan. All the four assays provide precised, accurate and comparable results. However, some differences were observed. For instance, TLC, HPLC and LC-MS/MS methodologies offered the advantage of the quantification of individual toxins in contrast to ELISA technique. The contamination ranges of AFB₁/AFB₂ as determined by TLC, HPLC and LC-MS/MS were 1.18-9.97/0.59-1.52, 0.16-10.54/0.26-1.35 and 0.11-10.88/0.38-1.48 µg/kg, respectively. However, AFG₁ and AFG₂ were not detected in any tested samples. Furthermore, owing to low-detection limit and sensitivity, HPLC and LC-MS/MS methodologies have identified greater number of contaminated samples in comparison to TLC and ELISA techniques. The overall average results of total AFs as provided by HPLC (3.79 µg/kg) and LC-MS/MS (3.89 µg/kg) were found higher in comparison to TLC (3.68 µg/kg) and ELISA (3.70 µg/kg). On the basis of achieved results, it was concluded that TLC, HPLC, LC-MS/MS and ELISA techniques are valuable tool for the quantification of AFs in cereals and grains. Furthermore, HPLC and LC-MS/MS techniques offer an added advantage for the detection of AFs in diminutive levels.

  11. Comparative Proteomic Profile of the Human Umbilical Cord Blood Exosomes between Normal and Preeclampsia Pregnancies with High-Resolution Mass Spectrometry


    Ruizhe Jia; Jingyun Li; Can Rui; Hui Ji; Hongjuan Ding; Yuanqing Lu; Wei De; Lizhou Sun


    Background/Aims: Exosomes are extracellular vesicles that are involved in several biological processes. The roles of proteins from human umbilical cord blood exosomes in the pathogenesis of preeclampsia remains poorly understood. Methods: In this study, we used high-resolution LC-MS/MS technologies to construct a comparative proteomic profiling of human umbilical cord blood exosomes between normal and preeclamptic pregnancies. Results: A total of 221 proteins were detected in human umbilical ...

  12. Automated DBS microsampling, microscale automation and microflow LC-MS for therapeutic protein PK. (United States)

    Zhang, Qian; Tomazela, Daniela; Vasicek, Lisa A; Spellman, Daniel S; Beaumont, Maribel; Shyong, BaoJen; Kenny, Jacqueline; Fauty, Scott; Fillgrove, Kerry; Harrelson, Jane; Bateman, Kevin P


    Reduce animal usage for discovery-stage PK studies for biologics programs using microsampling-based approaches and microscale LC-MS. We report the development of an automated DBS-based serial microsampling approach for studying the PK of therapeutic proteins in mice. Automated sample preparation and microflow LC-MS were used to enable assay miniaturization and improve overall assay throughput. Serial sampling of mice was possible over the full 21-day study period with the first six time points over 24 h being collected using automated DBS sample collection. Overall, this approach demonstrated comparable data to a previous study using single mice per time point liquid samples while reducing animal and compound requirements by 14-fold. Reduction in animals and drug material is enabled by the use of automated serial DBS microsampling for mice studies in discovery-stage studies of protein therapeutics.


    Directory of Open Access Journals (Sweden)

    Chi Chen


    Full Text Available Xenobiotic exposure, especially high-dose or repeated exposure of xenobiotics, can elicit detrimental effects on biological systems through diverse mechanisms. Changes in metabolic systems, including formation of reactive metabolites and disruption of endogenous metabolism, are not only the common consequences of toxic xenobiotic exposure, but in many cases are the major causes behind development of xenobiotic-induced toxicities (XIT. Therefore, examining the metabolic events associated with XIT generates mechanistic insights into the initiation and progression of XIT, and provides guidance for prevention and treatment. Traditional bioanalytical platforms that target only a few suspected metabolites are capable of validating the expected outcomes of xenobiotic exposure. However, these approaches lack the capacity to define global changes and to identify unexpected events in the metabolic system. Recent developments in high-throughput metabolomics have dramatically expanded the scope and potential of metabolite analysis. Among all analytical techniques adopted for metabolomics, liquid chromatography-mass spectrometry (LC-MS has been most widely used for metabolomic investigations of XIT due to its versatility and sensitivity in metabolite analysis. In this review, technical platform of LC-MS-based metabolomics, including experimental model, sample preparation, instrumentation, and data analysis, are discussed. Applications of LC-MS-based metabolomics in exploratory and hypothesis-driven investigations of XIT are illustrated by case studies of xenobiotic metabolism and endogenous metabolism associated with xenobiotic exposure.

  14. Quantitative LC-MS/MS Analysis of Proteins Involved in Metastasis of Breast Cancer (United States)

    Goto, Rieko; Nakamura, Yasushi; Takami, Tomonori; Sanke, Tokio; Tozuka, Zenzaburo


    The purpose of this study was to develop quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the analysis of proteins involved in metastasis of breast cancer for diagnosis and determining disease prognosis, as well as to further our understand of metastatic mechanisms. We have previously demonstrated that the protein type XIV collagen may be specifically expressed in metastatic tissues by two dimensional LC-MS/MS. In this study, we developed quantitative LC-MS/MS methods for type XIV collagen. Type XIV collagen was quantified by analyzing 2 peptides generated by digesting type XIV collagen using stable isotope-labeled peptides. The individual concentrations were equivalent between 2 different peptides of type XIV collagen by evaluation of imprecise transitions and using the best transition for the peptide concentration. The results indicated that type XIV collagen is highly expressed in metastatic tissues of patients with massive lymph node involvement compared to non-metastatic tissues. These findings were validated by quantitative real-time RT-PCR. Further studies on type XIV collagen are desired to verify its role as a prognostic factor and diagnosis marker for metastasis. PMID:26176947

  15. Lung Cancer Serum Biomarker Discovery Using Label Free LC-MS/MS (United States)

    Zeng, Xuemei; Hood, Brian L.; Zhao, Ting; Conrads, Thomas P.; Sun, Mai; Gopalakrishnan, Vanathi; Grover, Himanshu; Day, Roger S.; Weissfeld, Joel L.; Wilson, David O.; Siegfried, Jill M.; Bigbee, William L.


    Introduction Lung cancer remains the leading cause of cancer-related death with poor survival due to the late stage at which lung cancer is typically diagnosed. Given the clinical burden from lung cancer, and the relatively favorable survival associated with early stage lung cancer, biomarkers for early detection of lung cancer are of important potential clinical benefit. Methods We performed a global lung cancer serum biomarker discovery study using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a set of pooled non-small cell lung cancer (NSCLC) case sera and matched controls. Immunoaffinity subtraction was used to deplete the top most abundant serum proteins; the remaining serum proteins were subjected to trypsin digestion and analyzed in triplicate by LC-MS/MS. The tandem mass spectrum data were searched against the human proteome database and the resultant spectral counting data were used to estimate the relative abundance of proteins across the case/control serum pools. The spectral counting derived abundances of some candidate biomarker proteins were confirmed with multiple reaction monitoring MS assays. Results A list of 49 differentially abundant candidate proteins was compiled by applying a negative binomial regression model to the spectral counting data (pbiomarkers with statistically significant differential abundance across the lung cancer case/control pools which, when validated, could improve lung cancer early detection. PMID:21304412

  16. Direct identification of amyloids by label-free quantitative LC-MS

    DEFF Research Database (Denmark)

    Dueholm, Morten Simonsen; Danielsen, Heidi Nolsøe; Hansen, Susan Hove

    Direct identification of amyloids by label-free quantitative LC-MS H. N. Danielsen, S. H. Hansen, F.-A. Herbst, P. H. Nielsen, M. S. Dueholm Amyloids are highly ordered fibrillar protein polymers used by organisms from all domains of life due to their exceptional properties. We have previously...... that have been studied so far have already provided an astonishing demonstration of how the amyloids can be exploited with roles ranging structural components of biofilms, cell envelopes and spore coats to cytotoxins and as reservoirs for quorum-sensing signaling molecules. The identification of novel...... adhesive and therefore bind to pipette tips and other consumables. Pure cultures, large sample volumes and high productivity of amyloids are therefore required for successful purification. We here present a quantitative proteomics technique that allow direct identification of functional amyloid candidates...

  17. A new high-throughput LC-MS method for the analysis of complex fructan mixtures

    DEFF Research Database (Denmark)

    Verspreet, Joran; Hansen, Anders Holmgaard; Dornez, Emmie


    In this paper, a new liquid chromatography-mass spectrometry (LC-MS) method for the analysis of complex fructan mixtures is presented. In this method, columns with a trifunctional C18 alkyl stationary phase (T3) were used and their performance compared with that of a porous graphitized carbon (PGC...... of polymerization of 10. This indicates that wheat grain fructans do not, or not only, have a simple linear structure. The presented method paves the way for elucidation of fructan structures in complex mixtures that contain many structural isomers....

  18. Analysis of pesticide residues in strawberries and soils by GC-MS/MS, LC-MS/MS and two-dimensional GC– time-of-flight MS comparing organic and integrated pest management farming

    NARCIS (Netherlands)

    Fernandes, V.C.; Lehotay, S.J.; Geis-Asteggiantec, L.; Kwon, H.; Mol, J.G.J.; Kamp, van der H.J.; Mateus, N.; Domingues, V.F.; Delerue-Matos, C.


    In this study, we analyzed 22 strawberry and soil samples after their collection over the course of 2 years to compare the residue profiles from organic farming to integrated pest management practices in Portugal. For sample preparation, we used the citrate-buffered version of the quick, easy,

  19. Analysis of benzodiazepines and their metabolites using DBS cards and LC-MS/MS. (United States)

    Lee, Heesang; Park, Yujin; Jo, Jiyeong; In, Sangwhan; Park, Yonghoon; Kim, Eunmi; Pyo, Jaesung; Choe, Sanggil


    Dried Blood Spot (DBS) has been used a blood extraction method for inherited metabolic disorder screening since 1960s. With introduction of LC-MS/MS, not only DBS could be used to analysis drugs in small blood volume, but in various fields, such as toxicology, drug therapeutic monitoring, drug diagnostic screening, and illicit drugs. In toxicology field, many drugs (e.g. benzodiazepines, acetaminophen, small molecule drugs) have been tested with DBS. Compared with earlier blood extraction methods (SPE and LLE), DBS has lots of advantages; lower blood volume (less than 50μL), shorter analysis time caused by a more concise analysis procedure and lower cost. We optimized the DBS procedure and LC-MS/MS conditions for 18 benzodiazepines, seven benzodiazepine metabolites, and one z-drug (zolpidem) analysis in blood. 30μL of whole blood was spotted on FTA DMPK card C and dried for 2h in a desiccator. A 6-mm disk was punched and vortexed for 1min in a centrifuge tube with 300μL methanol/acetonitrile mixture (1:1, v/v). After evaporation, redissolved in 100μL mobile phase of LC-MS/MS and 5μL was injected. In the analysis for 26 target compounds in blood, all of the method validation parameters - LLOD, LLOQ, accuracy (intra- and inter-assay), and precision (intra- and inter-assay) - were satisfied with method validation criteria, within 15%. The results of matrix effect, recovery, and process efficiency were good. We developed a fast and reliable sample preparation method using DBS for 26 benzodiazepines, benzodiazepine metabolites, and z-drug (zolpidem). Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. LC-MS (/MS) in clinical toxicology screening methods. (United States)

    Viette, Véronique; Hochstrasser, Denis; Fathi, Marc


    Toxicological screening is the analysis of biological samples to detect and identify unknown compounds. The high selectivity and sensitivity of liquid chromatography (LC) coupled to mass spectrometry (MS) or tandem mass spectrometry (MS/MS) technology provide an attractive alternative to the current methods (LC-UV, GC/MS, etc.). For these reasons, an increasing number of applications are being published. This paper is a brief overview of LC-MS(/MS) screening methods developed for clinical toxicology in recent years. Various sample treatments, chromatographic separations and detection by mass spectrometry can be combined to obtain screening methods adapted to the constraints and needs of clinical toxicology laboratories. Currently the techniques are in the hands of specialists, mainly in academic institutions. However, the evolution in technology should allow application of these techniques as a tool in toxicology laboratories, thus allowing a more widespread exploitation of their potential.

  1. Simultaneous determination of three flavonoids and one coumarin by LC-MS/MS: application to a comparative pharmacokinetic study in normal and arthritic rats after oral administration of Daphne genkwa extract. (United States)

    Zhou, Luyi; Li, Jing; Yan, Chong


    A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for investigating the pharmacokinetics of umbelliferone, apigenin, genkwanin, and hydroxygenkwanin after oral administration of Daphne genkwa extract. Plasma samples were treated by protein precipitation with acetonitrile. Analytes were detected by triple-quadrupole MS/MS with an ESI source in negative selection reaction monitoring mode. The transitions of m/z 161→133 for umbelliferone, m/z 269→117 for apigenin, m/z 283→268 for genkwanin, and m/z 299→284 for hydroxygenkwanin were confirmed for quantification. Chromatographic separation was conducted using an Eclipse XDB-C 18 column, and the applied isocratic elution program allowed for simultaneous determination of the four analytes for a total run time of 2.5 min. The linearity was validated over the plasma concentration ranges of 1.421-1421 ng/mL for umbelliferone, 0.845-845 ng/mL for apigenin, 1.025-1025 ng/mL for genkwanin, and 0.845-845 ng/mL for hydroxygenkwanin. The extraction recovery rate was >82.7% for each analyte. No apparent matrix effect was observed during the bioanalysis. After full validation, the proposed method was successfully applied to compare the pharmacokinetics of these analytes between normal and arthritic rats. This article is protected by copyright. All rights reserved.

  2. Software and Database Usage on Metabolomic Studies: Using XCMS on LC-MS Data Analysis

    Directory of Open Access Journals (Sweden)

    Mustafa Celebier


    Full Text Available Metabolome is the complete set of small-molecule metabolites to be found in a cell or a single organism. Metabolomics is the scientific study to determine and identify the chemicals in metabolome with advanced analytical techniques. Nowadays, the elucidation of the molecular mechanism of any disease with genome analysis and proteome analysis is not sufficient. Instead of these, a holistic assessment including metabolomic studies provides rational and accurate results. Metabolite levels in an organism are associated with the cellular functions. Thus, determination of the metabolite amounts identifies the phenotype of a cell or tissue related with the genetic and some other variations. Even though, the analysis of metabolites for medical diagnosis and therapy have been performed for a long time, the studies to improve the analysis methods for metabolite profiling are recently increased. The application of metabolomics includes the identification of biomarkers, enzyme-substract interactions, drug-activity studies, metabolic pathway analysis and some other studies related with the system biology. The preprocessing and computing of the data obtained from LC-MS, GC-MS, CE-MS and NMR for metabolite profiling are helpful for preventing from time consuming manual data analysis processes and possible random errors on profiling period. In addition, such preprocesses allow us to identify low amount of metabolites which are not possible to be analyzed by manual processing. Therefore, the usage of software and databases for this purpose could not be ignored. In this study, it is briefly presented the software and database used on metabolomics and it is evaluated the capability of these software on metabolite profiling. Particularly, the performance of one of the most popular software called XCMS on the evaluation of LC-MS results for metabolomics was overviewed. In the near future, metabolomics with software and database support is estimated to be a routine

  3. Comparative shotgun proteomic analysis of Clostridium acetobutylicum from butanol fermentation using glucose and xylose

    Directory of Open Access Journals (Sweden)

    Verberkmoes Nathan C


    Full Text Available Abstract Background Butanol is a second generation biofuel produced by Clostridium acetobutylicum through acetone-butanol-ethanol (ABE fermentation process. Shotgun proteomics provides a direct approach to study the whole proteome of an organism in depth. This paper focuses on shotgun proteomic profiling of C. acetobutylicum from ABE fermentation using glucose and xylose to understand the functional mechanisms of C. acetobutylicum proteins involved in butanol production. Results We identified 894 different proteins in C. acetobutylicum from ABE fermentation process by two dimensional - liquid chromatography - tandem mass spectrometry (2D-LC-MS/MS method. This includes 717 proteins from glucose and 826 proteins from the xylose substrate. A total of 649 proteins were found to be common and 22 significantly differentially expressed proteins were identified between glucose and xylose substrates. Conclusion Our results demonstrate that flagellar proteins are highly up-regulated with glucose compared to xylose substrate during ABE fermentation. Chemotactic activity was also found to be lost with the xylose substrate due to the absence of CheW and CheV proteins. This is the first report on the shotgun proteomic analysis of C. acetobutylicum ATCC 824 in ABE fermentation between glucose and xylose substrate from a single time data point and the number of proteins identified here is more than any other study performed on this organism up to this report.

  4. Evaluation of batch effect elimination using quality control replicates in LC-MS metabolite profiling. (United States)

    Sánchez-Illana, Ángel; Piñeiro-Ramos, Jose David; Sanjuan-Herráez, Juan Daniel; Vento, Máximo; Quintás, Guillermo; Kuligowski, Julia


    Systematic variation of the instrument's response both within- and between-batches is frequently observed in untarget LC-MS metabolomics involving the analysis of a large number of samples. The so-called batch effect decreases the statistical power and has a negative impact on repeatability and reproducibility of the results. As there is no standard way of assessing or correcting LC-MS batch effects and there is no single method providing optimal results in all situations, the selection of the optimal approach is not trivial. This work explores the effectiveness of a set of tools for batch effect assessment. Qualitative tools include the monitoring of spiked internal standards, principal component analysis and hierarchical cluster analysis. Quantitative tools comprise the distribution of RSD QC values, the median Pearson correlation coefficient in QCs, the ratio of random features in QCs using the runs test, as well as multivariate tools such as the δ-statistic, Silhouette plots, Principal Variance Component Analysis and the expected technical variation in the prediction. Results show that qualitative and quantitative approaches are complementary and that by limiting the analysis to QCs the power to detect and evaluate both within and between batch effects is increased. Besides, the graphical integration of outputs from multiple quantitative tools facilitates the evaluation of batch effects and it is proposed as a straightforward way for comparing and tailoring batch effect elimination approaches. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Plasma metabolic profiling of dairy cows affected with clinical ketosis using LC/MS technology. (United States)

    Li, Y; Xu, C; Xia, C; Zhang, Hy; Sun, Lw; Gao, Y


    Ketosis in dairy cattle is an important metabolic disorder. Currently, the plasma metabolic profile of ketosis as determined using liquid chromatography-mass spectrometry (LC/MS) has not been reported. To investigate plasma metabolic profiles from cows with clinical ketosis in comparison to control cows. Twenty Holstein dairy cows were divided into two groups based on clinical signs and plasma β-hydroxybutyric acid and glucose concentrations 7-21 days postpartum: clinical ketosis and control cows. Plasma metabolic profiles were analyzed using LC/MS. Data were processed using principal component analysis and orthogonal partial least-squares discriminant analysis. Compared to control cows, the levels of valine, glycine, glycocholic, tetradecenoic acid, and palmitoleic acid increased significantly in clinical ketosis. On the other hand, the levels of arginine, aminobutyric acid, leucine/isoleucine, tryptophan, creatinine, lysine, norcotinine, and undecanoic acid decreased markedly. Our results showed that the metabolic changes in cows with clinical ketosis involve complex metabolic networks and signal transduction. These results are important for future studies elucidating the pathogenesis, diagnosis, and prevention of clinical ketosis in dairy cows.

  6. Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples. (United States)

    Fang, Nianbai; Yu, Shanggong; Ronis, Martin Jj; Badger, Thomas M


    High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-peak with reliable retention time (Rt.). However, in the current study, we have found that under the same LC-MS conditions, the Rt. and shape of LC-peaks of bile acids in urine samples from animals fed dissimilar diets differed significantly among each other. To verify this matrix effect, 17 authentic bile acid standards were dissolved in pure methanol or in methanol containing extracts of urine from pigs consuming either breast milk or infant formula and analyzed by LC-MS/MS. The matrix components in urine from piglets fed formula significantly reduced the LC-peak Rt. and areas of bile acids. This is the first characterization of this matrix effect on Rt. in the literature. Moreover, the matrix effect resulted in an unexpected LC behavior: one single compound yielded two LC-peaks, which broke the rule of one LC-peak for one compound. The three bile acid standards which exhibited this unconventional LC behavior were chenodeoxycholic acid, deoxycholic acid, and glycocholic acid. One possible explanation for this effect is that some matrix components may have loosely bonded to analytes, which changed the time analytes were retained on a chromatography column and interfered with the ionization of analytes in the MS ion source to alter the peak area. This study indicates that a comprehensive understanding of matrix effects is needed towards improving the use of HPLC and LC-MS/MS techniques for qualitative and quantitative analyses of analytes in pharmacokinetics, proteomics/metabolomics, drug development, and sports drug testing, especially when LC-MS/MS data are analyzed by automation software where identification of an analyte is based on its exact molecular weight and Rt

  7. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity

    Directory of Open Access Journals (Sweden)

    Zulezwan A. Malik


    Full Text Available Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC coupled to mass spectrometry (MS affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient (i.e., 3 h per sample proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group bred as either high- or low-capacity runners (HCR and LCR, respectively that exhibited a 6.4-fold difference (1,625 ± 112 m vs. 252 ± 43 m, p < 0.0001 in running capacity during a standardized treadmill test. Soluble muscle proteins were extracted, digested with trypsin and individual biological replicates (50 ng of tryptic peptides subjected to LC-MS profiling. Proteins were identified by triplicate LC-MS/MS analysis of a pooled sample of each biological replicate. Differential expression profiling was performed on relative abundances (RA of parent ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897 and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5. Sixteen proteins were significantly (p < 0.05 more abundant in HCR muscle and hierarchal clustering of the profiling data highlighted two protein subgroups, which encompassed proteins associated with either the respiratory chain or fatty acid oxidation. Heart-type fatty acid binding protein (FABPH was 1

  8. LC-MS-Based Metabolic Fingerprinting of Aqueous Humor

    Directory of Open Access Journals (Sweden)

    Karolina Pietrowska


    Full Text Available Aqueous humor (AH is a transparent fluid which fills the anterior and posterior chambers of the eye. It supplies nutrients and removes metabolic waste from avascular tissues in the eye. Proper homeostasis of AH is required to maintain adequate intraocular pressure as well as optical and refractive properties of the eye. Application of metabolomics to study human AH may improve knowledge about the molecular mechanisms of eye diseases. Until now, global analysis of metabolites in AH has been mainly performed using NMR. Among the analytical platforms used in metabolomics, LC-MS allows for the highest metabolome coverage. The aim of this study was to develop a method for extraction and analysis of AH metabolites by LC-QTOF-MS. Different protocols for AH preparation were tested. The best results were obtained when one volume of AH was mixed with one volume of methanol : ethanol (1 : 1. In the final method, 2 µL of extracted sample was analyzed by LC-QTOF-MS. The method allowed for reproducible measurement of over 1000 metabolic features. Almost 250 metabolites were identified in AH and assigned to 47 metabolic pathways. This method is suitable to study the potential role of amino acids, lipids, oxidative stress, or microbial metabolites in development of ocular diseases.

  9. On studying protein phosphorylation patterns using bottom-up LC-MS/MS: the case of human alpha-casein

    DEFF Research Database (Denmark)

    Kjeldsen, Frank; Savitski, Mikhail M; Nielsen, Michael L


    Most proteomics studies involving mapping post-translational modifications, such as the phosphorylation of serine and threonine, are performed today using the 'bottom-up' approach. This approach involves enzymatic cleavage of proteins, most often by trypsin, with subsequent nano-LC-MS/MS. The occ......Most proteomics studies involving mapping post-translational modifications, such as the phosphorylation of serine and threonine, are performed today using the 'bottom-up' approach. This approach involves enzymatic cleavage of proteins, most often by trypsin, with subsequent nano......-LC-MS/MS. The occupancy rates of phosphosites in proteins may differ by orders of magnitude, and thus the occupancy rate must be reported for each occupied phosphosite. To highlight potential pitfalls in quantifying the occupancy rates, alpha(s1)-casein from human milk was selected as a model molecule representing...... moderately phosphorylated proteins. For this purpose, human milk from one Caucasian woman in the eighth month of lactation was used. The phosphorylation level of caseins is believed to have major implications for the formation of micelles that are involved in delivering valuable calcium phosphate and other...

  10. Enrichment and identification of integral membrane proteins from barley aleurone layers by reversed-phase chromatography, SDS-PAGE and LC-MS/MS

    DEFF Research Database (Denmark)

    Hynek, Radovan; Svensson, Birte; Nørregaard Jensen, Ole


    was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral......+-ATPase, two proteins possibly involved in ion-channel regulation and two proteins of unknown function. This represents the first analysis of membrane proteins involved in seed germination, using a proteomics approach....

  11. Simple strategies to enhance discovery of acetylation post-translational modifications by quadrupole-orbitrap LC-MS/MS. (United States)

    Manning, Andrew J; Lee, Jiyoung; Wolfgeher, Donald J; Kron, Stephen J; Greenberg, Jean T


    Enzyme-dependent post-translational modifications (PTMs) mediate the cellular regulation of proteins and can be discovered using proteomics. However, even where the peptides of interest can be enriched for analysis with state-of-the-art LC-MS/MS tools and informatics, only a fraction of peptide ions can be identified confidently. Thus, many PTM sites remain undiscovered and unconfirmed. In this minireview, we use a case study to discuss how the use of inclusion lists, turning off isotopic exclusion, and manual validation significantly increased depth of coverage, facilitating discovery of acetylation sites in targets of an acetyltransferase virulence factor. These underutilized strategies have the potential to help answer many mechanistic biological questions that large-scale proteomic studies cannot. Copyright © 2017. Published by Elsevier B.V.

  12. Quantitative proteomics analysis using 2D-PAGE to investigate the effects of cigarette smoke and aerosol of a prototypic modified risk tobacco product on the lung proteome in C57BL/6 mice. (United States)

    Elamin, Ashraf; Titz, Bjoern; Dijon, Sophie; Merg, Celine; Geertz, Marcel; Schneider, Thomas; Martin, Florian; Schlage, Walter K; Frentzel, Stefan; Talamo, Fabio; Phillips, Blaine; Veljkovic, Emilija; Ivanov, Nikolai V; Vanscheeuwijck, Patrick; Peitsch, Manuel C; Hoeng, Julia


    Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations. Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating

  13. Tryptic peptide screening for primary immunodeficiency disease by LC/MS-MS. (United States)

    Kerfoot, Sandra A; Jung, Sunhee; Golob, Karin; Torgerson, Troy R; Hahn, Si Houn


    Early diagnosis of primary immunodeficiency disorders (PIDDs) is critical for maximizing patient survival and clinical outcomes. Consequently, there is significant interest in developing broad-based, high-throughput, screening approaches capable of utilizing small blood volumes to identify patients with PIDD. We developed a novel proteomic screening approach using tandem mass spectrometry to simultaneously identify specific signature peptides derived from the transmembrane protein cluster of differentiation 3 (CD3)ɛ and the intracellular proteins Wiskott-Aldrich syndrome protein (WASP) and Bruton's tyrosine kinase (BTK) as markers of three life-threatening PIDDs; severe combined immunodeficiency, Wiskott-Aldrich syndrome, and X-linked Agammaglobulinemia. Signature peptides were analyzed by LC/MS-MS in proteolytically digested lysates from cell lines and white blood cells (WBCs). The amount of each peptide was determined by the ratio of the signature peptide peak area to that of a known amount of labeled standard peptide. Peptide concentrations were normalized to actin. We show that signature peptides from CD3ɛ, WASP, and BTK were readily detected in proteolytically digested cell lysate and their absence could correctly identify PIDD patients. This proof of concept study demonstrates the applicability of this approach to screen for PIDD and raises the possibility that it could be further multiplexed to identify additional PIDDs and potentially other disorders. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Spectral Counting Approach to Measure Selectivity of High-Resolution LC-MS Methods for Environmental Analysis. (United States)

    Renaud, Justin B; Sabourin, Lyne; Topp, Edward; Sumarah, Mark W


    Advances in high-resolution mass spectrometers have allowed for the development of nontargeted screening methods, where data sets can be archived and retrospectively mined as new environmental contaminants are identified. We have developed a spectral counting approach to calculate the selectivities of LC-MS acquisition modes taking mass accuracy, sample matrix, and the analyte properties into account. The selectivities of high-resolution MS (HRMS) alone or in combination with all-ion-fragmentation (AIF), data-independent-acquisition (DIA), and data-dependent-acquisition (DDA) modes, performed on a Q-Exactive Orbitrap were compared by retrospectively screening surface water samples for 95 pharmaceuticals. Samples were reanalyzed using targeted LC-MS/MS to confirm the accuracy of each acquisition method and to quantitate the 29 putatively detected drugs. LC-HRMS provided the lowest calculated selectivities and accordingly produced the highest number of false positives (6). In contrast, DDA provided the highest selectivities, yielding only one false positive; however, it was bias toward the most intense signals resulting in the detection of only 10 compounds. AIF had lower selectivities than traditional LC-MS/MS, produced one false positive and did not detect 6 confirmed compounds. Because of the high-quality archived data, DIA selectivities were better than traditional LC-MS/MS, showed no bias toward the most intense signals, achieved low limits of detection, and confidently detected the greatest number of pharmaceuticals (22) with only one false positive. This spectral counting method can be used across different instrument platforms or samples and provides a robust and empirical estimation of selectivities to give more confident detection of trace analytes.

  15. Fast targeted analysis of 132 acidic and neutral drugs and poisons in whole blood using LC-MS/MS. (United States)

    Di Rago, Matthew; Saar, Eva; Rodda, Luke N; Turfus, Sophie; Kotsos, Alex; Gerostamoulos, Dimitri; Drummer, Olaf H


    The aim of this study was to develop an LC-MS/MS based screening technique that covers a broad range of acidic and neutral drugs and poisons by combining a small sample volume and efficient extraction technique with simple automated data processing. After protein precipitation of 100μL of whole blood, 132 common acidic and neutral drugs and poisons including non-steroidal anti-inflammatory drugs, barbiturates, anticonvulsants, antidiabetics, muscle relaxants, diuretics and superwarfarin rodenticides (47 quantitated, 85 reported as detected) were separated using a Shimadzu Prominence HPLC system with a C18 separation column (Kinetex XB-C18, 4.6mm×150mm, 5μm), using gradient elution with a mobile phase of 25mM ammonium acetate buffer (pH 7.5)/acetonitrile. The drugs were detected using an ABSciex(®) API 2000 LC-MS/MS system (ESI+ and -, MRM mode, two transitions per analyte). The method was fully validated in accordance with international guidelines. Quantification data obtained using one-point calibration compared favorably to that using multiple calibrants. The presented LC-MS/MS assay has proven to be applicable for determination of the analytes in blood. The fast and reliable extraction method combined with automated processing gives the opportunity for high throughput and fast turnaround times for forensic and clinical toxicology. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. LC-MS analysis of the plasma metabolome–a novel sample preparation strategy

    DEFF Research Database (Denmark)

    Skov, Kasper; Hadrup, Niels; Smedsgaard, Jørn


    Blood plasma is a well-known body fluid often analyzed in studies on the effects of toxic compounds as physiological or chemical induced changes in the mammalian body are reflected in the plasma metabolome. Sample preparation prior to LC-MS based analysis of the plasma metabolome is a challenge...... as plasma contains compounds with very different properties. Besides, proteins, which usually are precipitated with organic solvent, phospholipids, are known to cause ion suppression in electrospray mass spectrometry. We have compared two different sample preparation techniques prior to LC-qTOF analysis...... of plasma samples: The first is protein precipitation; the second is protein precipitation followed by solid phase extraction with sub-fractionation into three sub-samples; a phospholipid, a lipid and a polar sub-fraction. Molecular feature extraction of the data files from LC-qTOF analysis of the samples...

  17. A Critical Assessment of Feature Selection Methods for Biomarker Discovery in Clinical Proteomics

    NARCIS (Netherlands)

    Christin, Christin; Hoefsloot, Huub C. J.; Smilde, Age K.; Hoekman, B.; Suits, Frank; Bischoff, Rainer; Horvatovich, Peter

    In this paper, we compare the performance of six different feature selection methods for LC-MS-based proteomics and metabolomics biomarker discovery-t test, the Mann-Whitney-Wilcoxon test (mww test), nearest shrunken centroid (NSC), linear support vector machine-recursive features elimination

  18. QC metrics from CPTAC raw LC-MS/MS data interpreted through multivariate statistics. (United States)

    Wang, Xia; Chambers, Matthew C; Vega-Montoto, Lorenzo J; Bunk, David M; Stein, Stephen E; Tabb, David L


    Shotgun proteomics experiments integrate a complex sequence of processes, any of which can introduce variability. Quality metrics computed from LC-MS/MS data have relied upon identifying MS/MS scans, but a new mode for the QuaMeter software produces metrics that are independent of identifications. Rather than evaluating each metric independently, we have created a robust multivariate statistical toolkit that accommodates the correlation structure of these metrics and allows for hierarchical relationships among data sets. The framework enables visualization and structural assessment of variability. Study 1 for the Clinical Proteomics Technology Assessment for Cancer (CPTAC), which analyzed three replicates of two common samples at each of two time points among 23 mass spectrometers in nine laboratories, provided the data to demonstrate this framework, and CPTAC Study 5 provided data from complex lysates under Standard Operating Procedures (SOPs) to complement these findings. Identification-independent quality metrics enabled the differentiation of sites and run-times through robust principal components analysis and subsequent factor analysis. Dissimilarity metrics revealed outliers in performance, and a nested ANOVA model revealed the extent to which all metrics or individual metrics were impacted by mass spectrometer and run time. Study 5 data revealed that even when SOPs have been applied, instrument-dependent variability remains prominent, although it may be reduced, while within-site variability is reduced significantly. Finally, identification-independent quality metrics were shown to be predictive of identification sensitivity in these data sets. QuaMeter and the associated multivariate framework are available from and , respectively.

  19. Multidimensional protein fractionation of blood proteins coupled to data-independent nanoLC-MS/MS analysis. (United States)

    Levin, Yishai; Jaros, Julian A J; Schwarz, Emanuel; Bahn, Sabine


    In order to exploit human blood as a source of protein disease biomarkers, robust analytical methods are needed to overcome the inherent molecular complexity of this bio-fluid. We present the coupling of label-free SAX chromatography and IMAC to a data-independent nanoLC-MS/MS (nanoLC-MS(E)) platform for analysis of blood plasma and serum proteins. The methods were evaluated using protein standards added at different concentrations to two groups of samples. The results demonstrate that both techniques enable accurate protein quantitation using low sample volumes and a minimal number of fractions. Combining both methods, 883 unique proteins were identified, of which 423 proteins showed high reproducibility. The two approaches resulted in identification of unique molecular signatures with an overlap of approximately 30%, thus providing complimentary information on sub-proteomes. These methods are potentially useful for systems biology, biomarker discovery, and investigation of phosphoproteins in blood. (c) 2009 Elsevier B.V. All rights reserved.

  20. Comparative mitochondrial proteomics: perspective in human diseases

    Directory of Open Access Journals (Sweden)

    Jiang Yujie


    Full Text Available Abstract Mitochondria are the most complex and the most important organelles of eukaryotic cells, which are involved in many cellular processes, including energy metabolism, apoptosis, and aging. And mitochondria have been identified as the "hot spot" by researchers for exploring relevant associated dysfunctions in many fields. The emergence of comparative proteomics enables us to have a close look at the mitochondrial proteome in a comprehensive and effective manner under various conditions and cellular circumstances. Two-dimensional electrophoresis combined with mass spectrometry is still the most popular techniques to study comparative mitochondrial proteomics. Furthermore, many new techniques, such as ICAT, MudPIT, and SILAC, equip researchers with more flexibilities inselecting proper methods. This article also reviews the recent development of comparative mitochondrial proteomics on diverse human diseases. And the results of mitochondrial proteomics enhance a better understanding of the pathogenesis associated with mitochondria and provide promising therapeutic targets.

  1. Label-Free LC-MS Profiling of Skeletal Muscle Reveals Heart-Type Fatty Acid Binding Protein as a Candidate Biomarker of Aerobic Capacity. (United States)

    Malik, Zulezwan Ab; Cobley, James N; Morton, James P; Close, Graeme L; Edwards, Ben J; Koch, Lauren G; Britton, Steven L; Burniston, Jatin G


    Two-dimensional gel electrophoresis provides robust comparative analysis of skeletal muscle, but this technique is laborious and limited by its inability to resolve all proteins. In contrast, orthogonal separation by SDS-PAGE and reverse-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) affords deep mining of the muscle proteome, but differential analysis between samples is challenging due to the greater level of fractionation and the complexities of quantifying proteins based on the abundances of their tryptic peptides. Here we report simple, semi-automated and time efficient ( i.e ., 3 h per sample) proteome profiling of skeletal muscle by 1-dimensional RPLC electrospray ionisation tandem MS. Solei were analysed from rats (n = 5, in each group) bred as either high- or low-capacity runners (HCR and LCR, respectively) that exhibited a 6.4-fold difference (1,625 ± 112 m vs . 252 ± 43 m, p ions, which spanned three orders of magnitude. In total, 207 proteins were analysed, which encompassed almost all enzymes of the major metabolic pathways in skeletal muscle. The most abundant protein detected was type I myosin heavy chain (RA = 5,843 ± 897) and the least abundant protein detected was heat shock 70 kDa protein (RA = 2 ± 0.5). Sixteen proteins were significantly ( p ion (551.21 m/z ) of the doubly-charged peptide SLGVGFATR (454.19 m/z ) of residues 23-31 of FABPH. SRM was conducted on technical replicates of each biological sample and exhibited a coefficient of variation of 20%. The abundance of FABPH measured by SRM was 2.84-fold greater ( p = 0.0095) in HCR muscle. In addition, SRM of FABPH was performed in vastus lateralis samples of young and elderly humans with different habitual activity levels (collected during a previous study) finding FABPH abundance was 2.23-fold greater ( p = 0.0396) in endurance-trained individuals regardless of differences in age. In summary, our findings in HCR/LCR rats provide protein-level confirmation for

  2. A Rapid and Sensitive LC-MS/MS Method for the Determination of ...

    African Journals Online (AJOL)

    VIS) detection of the analytes is also reported. Sulfonamides exhibited higher response factors towards UV/VIS than mass spectrometer detection and the opposite was true for the rest of the analytes. KEYWORDS: Quadrupole ion trap, LC-MS, ...

  3. Clustering with position-specific constraints on variance: Applying redescending M-estimators to label-free LC-MS data analysis

    Directory of Open Access Journals (Sweden)

    Mani D R


    Full Text Available Abstract Background Clustering is a widely applicable pattern recognition method for discovering groups of similar observations in data. While there are a large variety of clustering algorithms, very few of these can enforce constraints on the variation of attributes for data points included in a given cluster. In particular, a clustering algorithm that can limit variation within a cluster according to that cluster's position (centroid location can produce effective and optimal results in many important applications ranging from clustering of silicon pixels or calorimeter cells in high-energy physics to label-free liquid chromatography based mass spectrometry (LC-MS data analysis in proteomics and metabolomics. Results We present MEDEA (M-Estimator with DEterministic Annealing, an M-estimator based, new unsupervised algorithm that is designed to enforce position-specific constraints on variance during the clustering process. The utility of MEDEA is demonstrated by applying it to the problem of "peak matching"--identifying the common LC-MS peaks across multiple samples--in proteomic biomarker discovery. Using real-life datasets, we show that MEDEA not only outperforms current state-of-the-art model-based clustering methods, but also results in an implementation that is significantly more efficient, and hence applicable to much larger LC-MS data sets. Conclusions MEDEA is an effective and efficient solution to the problem of peak matching in label-free LC-MS data. The program implementing the MEDEA algorithm, including datasets, clustering results, and supplementary information is available from the author website at

  4. Diet-induced perturbation of the rat liver mitochondrial acetylome studied by quantitative (iTRAQ) LC-MS/MS

    DEFF Research Database (Denmark)

    León, Ileana R.; Schwämmle, Veit; Williamson, James

    -acetylation of mitochondrial proteins has emerged as a key regulator of cellular metabolism. The acetylated proteome includes enzymes involved in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism and glycogen metabolism. A mammal’s inability to handle excess energy intake...... levels1, 2. Methods A rat liver mitochondria-enriched sample was used to establish an iTRAQ-based LC-MS/MS strategy for determination of acetylated proteins. Immuno-affinity enrichment of acetylated peptides was carried out with anti-acetyl Lysine antibody (Immune Pharmaceuticals), as previously...... acetylation targets protein complexes and co-regulates major cellular functions. Science 2009, 325, (5942), 834-40....

  5. Development and Validation of an LC-MS/MS Method and Comparison with a GC-MS Method to Measure Phenytoin in Human Brain Dialysate, Blood, and Saliva

    Directory of Open Access Journals (Sweden)

    Raphael Hösli


    Full Text Available Phenytoin (PHT is one of the most often used critical dose drugs, where insufficient or excessive dosing can have severe consequences such as seizures or toxicity. Thus, the monitoring and precise measuring of PHT concentrations in patients is crucial. This study develops and validates an LC-MS/MS method for the measurement of phenytoin concentrations in different body compartments (i.e., human brain dialysate, blood, and saliva and compares it with a formerly developed GC-MS method that measures PHT in the same biological matrices. The two methods are evaluated and compared based on their analytical performance, appropriateness to analyze human biological samples, including corresponding extraction and cleanup procedures, and their validation according to ISO 17025/FDA Guidance for Industry. The LC-MS/MS method showed a higher performance compared with the GC-MS method. The LC-MS/MS was more sensitive, needed a smaller sample volume (25 µL and less chemicals, was less time consuming (cleaning up, sample preparation, and analysis, and resulted in a better LOD ( 0.995 for all tested matrices (blood, saliva, and dialysate. For larger sample numbers as in pharmacokinetic/pharmacodynamic studies and for bedside as well as routine analyses, the LC-MS/MS method offers significant advantages over the GC-MS method.

  6. Using a spike-in experiment to evaluate analysis of LC-MS data

    Directory of Open Access Journals (Sweden)

    Tuli Leepika


    Full Text Available Abstract Background Recent advances in liquid chromatography-mass spectrometry (LC-MS technology have led to more effective approaches for measuring changes in peptide/protein abundances in biological samples. Label-free LC-MS methods have been used for extraction of quantitative information and for detection of differentially abundant peptides/proteins. However, difference detection by analysis of data derived from label-free LC-MS methods requires various preprocessing steps including filtering, baseline correction, peak detection, alignment, and normalization. Although several specialized tools have been developed to analyze LC-MS data, determining the most appropriate computational pipeline remains challenging partly due to lack of established gold standards. Results The work in this paper is an initial study to develop a simple model with "presence" or "absence" condition using spike-in experiments and to be able to identify these "true differences" using available software tools. In addition to the preprocessing pipelines, choosing appropriate statistical tests and determining critical values are important. We observe that individual statistical tests could lead to different results due to different assumptions and employed metrics. It is therefore preferable to incorporate several statistical tests for either exploration or confirmation purpose. Conclusions The LC-MS data from our spike-in experiment can be used for developing and optimizing LC-MS data preprocessing algorithms and to evaluate workflows implemented in existing software tools. Our current work is a stepping stone towards optimizing LC-MS data acquisition and testing the accuracy and validity of computational tools for difference detection in future studies that will be focused on spiking peptides of diverse physicochemical properties in different concentrations to better represent biomarker discovery of differentially abundant peptides/proteins.

  7. Citrinin Determination in Red Fermented Rice Products by Optimized Extraction Method Coupled to Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS). (United States)

    Ji, Xiaofeng; Xu, Junfeng; Wang, Xiaofu; Qi, Peipei; Wei, Wei; Chen, Xiaoyun; Li, Rui; Zhou, Yu


    A rapid and sensitive method was developed and validated for citrinin determination in red fermented rice products by liquid chromatography tandem mass spectrometry (LC-MS/MS) under the selected reaction monitoring mode. Sample preparation was especially focused, and the quantitative methods of LC-MS/MS and high-performance liquid chromatography with fluorescence detection (HPLC-FLD) were compared. In red fermented rice samples, the limit of detection was 1.0 μg/kg for LC-MS/MS compared to 250 μg/kg for HPLC-FLD, the limit of quantification was 3.0 μg/kg for LC-MS/MS compared to 825 μg/kg for HPLC-FLD. High correlation coefficient was obtained (R(2) = 0.999) within the linear range (0.1 to 100 μg/L) in the MS method. The recoveries ranging from 80.9% to 106.5% were obtained in different spiking concentrations. The average intra- and inter-day accuracy ranged from 75.4% to 103.1%, and the intra- and inter-day precisions were from 3.3% to 7.9%. The developed method was applied to 12 commercial red fermented rice products, and citrinin was found in 10 samples ranging from 0.14 to 44.24 mg/kg. Compared to traditional qualitative and quantitative methods, the newly developed LC-MS/MS method for citrinin determination includes the merits of using a small amount of extraction solvent, simple preparation steps, and high sensitivity. © 2015 Institute of Food Technologists®

  8. Targeted and non-targeted detection of lemon juice adulteration by LC-MS and chemometrics. (United States)

    Wang, Zhengfang; Jablonski, Joseph E


    Economically motivated adulteration (EMA) of lemon juice was detected by LC-MS and principal component analysis (PCA). Twenty-two batches of freshly squeezed lemon juice were adulterated by adding an aqueous solution containing 5% citric acid and 6% sucrose to pure lemon juice to obtain 30%, 60% and 100% lemon juice samples. Their total titratable acidities, °Brix and pH values were measured, and then all the lemon juice samples were subject to LC-MS analysis. Concentrations of hesperidin and eriocitrin, major phenolic components of lemon juice, were quantified. The PCA score plots for LC-MS datasets were used to preview the classification of pure and adulterated lemon juice samples. Results showed a large inherent variability in the chemical properties among 22 batches of 100% lemon juice samples. Measurement or quantitation of one or several chemical properties (targeted detection) was not effective in detecting lemon juice adulteration. However, by using the LC-MS datasets, including both chromatographic and mass spectrometric information, 100% lemon juice samples were successfully differentiated from adulterated samples containing 30% lemon juice in the PCA score plot. LC-MS coupled with chemometric analysis can be a complement to existing methods for detecting juice adulteration.

  9. Simultaneous analysis of 17 diuretics in dietary supplements by HPLC and LC-MS/MS. (United States)

    Woo, H; Kim, J W; Han, K M; Lee, J H; Hwang, I S; Lee, J H; Kim, J; Kweon, S J; Cho, S; Chae, K R; Han, S Y; Kim, J


    In order to test health foods for illegally added diuretics for weight loss, we developed simple, rapid, selective, and sensitive methods using HPLC and LC-MS/MS for the simultaneous analysis of 17 diuretics in dietary supplements. HPLC conditions were set with a Capcell-pak C18, using a mobile phase consisting of gradient conditions, UV detection at 254 nm and validated for linearity (r(2)> 0.999), precision (CV ≤ 3%), recoveries (90.4-102.8%) and reproducibility. Identification and quantification of 17 diuretics were accomplished by ion-spray LC-MS/MS using multiple reaction monitoring (MRM). The chromatographic separation was carried out under the reversed-phase mechanism on an HSS-T3 column. The LC-MS/MS method was validated for linearity (r(2)> 0.99) and precision (CV Diuretics were not detected in all samples. Extraction recovery was also investigated and the extraction recoveries in different formulations were from 88% to 110% and from 81% to 116% using HPLC and LC-MS/MS, respectively. There was no significant difference in recoveries in the type of dietary supplements. Based on this result, the developed methods to monitor illegal drug adulterations in dietary supplements using HPLC and LC-MS/MS are simple, fast and reliable. Therefore, it is applicable to routine drug-adulteration screening.

  10. 2016 ASMS Workshop Review: Next Generation LC/MS: Critical Insights and Future Perspectives

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Hongying; Makarov, Alexander; Smith, Richard D.


    The pilot workshop on BNext Generation LC/MS: Critical Insights and Future Perspectives was held on the evening of June 6, 2016 at the 64th ASMS Conference on Mass Spectrometry and Allied Topics held in San Antonio, TX. The workshop, chaired by Hongying Gao (Pfizer), consisted of stimulating talks from distinguished speakers and open discussion among the audience and invited presenters.The objectives of this workshop were to better understand the advances and limitations of current technologies; to exchange perspectives on the next generation LC/MS; and to discuss/debate the features of next generation LC/MS focusing on the following three questions: (1) What would the next generation LC/MS look like? (2) How would it change the way we do analysis? and (3) What fundamental issues need to be resolved? A real-world case in the biopharmaceutical industry was presented by Hongying Gao on the needs by industry for LC/MS innovation and technology advancements. The primary invited speakers were Alexander Makarov (Thermo Fisher Scientific) and Richard (Dick) Smith (Pacific Northwest National Laboratory). The open discussions started with Q&A and comments for Alexander Makarov and Dick Smith, followed by insights and perspectives from members of the audience and other invited presenters who shared their thoughts addressing the above questions.

  11. Comparative proteomics of hydatid fluids from two Echinococcus multilocularis isolates. (United States)

    Monteiro, Karina M; Lorenzatto, Karina R; de Lima, Jeferson C; Dos Santos, Guilherme B; Förster, Sabine; Paludo, Gabriela P; Carvalho, Paulo C; Brehm, Klaus; Ferreira, Henrique B


    The hydatid fluid (HF) that fills Echinococcus multilocularis metacestode vesicles is a complex mixture of proteins from both parasite and host origin. Here, a LC-MS/MS approach was used to compare the HF composition of E. multilocularis H95 and G8065 isolates (EmH95 and EmG8065, respectively), which present differences in terms of growth and fertility. Overall, 446 unique proteins were identified, 392 of which (88%) were from parasite origin and 54 from culture medium. At least 256 of parasite proteins were sample exclusive, and 82 of the 136 shared proteins presented differential abundance between E. multilocularis isolates. The parasite's protein repertoires in EmH95 and EmG8065 HF samples presented qualitative and quantitative differences involving antigens, signaling proteins, proteolytic enzymes, protease inhibitors and chaperones, highlighting intraspecific singularities that could be correlated to biological features of each isolate. The repertoire of medium proteins found in the HF was also differential between isolates, and the relevance of the HF exogenous protein content for the parasite's biology is discussed. The repertoires of identified proteins also provided potential molecular markers for important biological features, such as parasite growth rate and fertility, as well potential protein targets for the development of novel diagnostic and treatment strategies for alveolar echinococcosis. E. multilocularis metacestode infection of mammal hosts involve complex interactions mediated by excretory/secretory (ES) products. The hydatid fluid (HF) that fills the E. multilocularis metacestode vesicles contains complex repertoires of parasite ES products and host proteins that mediate important molecular interactions determinant for parasite survival and development, and, consequently, to the infection outcome. HF has been also extensively reported as the main source of proteins for the immunodiagnosis of echinococcosis. The performed proteomic analysis

  12. Analysis of thyroid hormones in serum of Baikal seals and humans by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and immunoassay methods: application of the LC-MS/MS method to wildlife tissues. (United States)

    Kunisue, Tatsuya; Eguchi, Akifumi; Iwata, Hisato; Tanabe, Shinsuke; Kannan, Kurunthachalam


    Thyroid hormones (THs) are essential for the regulation of growth and development in both humans and wildlife. Until recently, TH concentrations in the tissues of animals have been examined by immunoassay (IA) methods. IA methods are sensitive, but for TH analysis, they are compromised by a lack of adequate specificity. In this study, we determined the concentrations of six THs, L-thyroxine (T(4)), 3,3',5-triiodo-L-thyronine (T(3)), 3,3',5'-triiodo-L-thyronine (rT(3)), 3,5-diiodo-L-thyronine (3,5-T(2)), 3,3'-diiodo-L-thyronine (3,3'-T(2)), and 3-iodo-L-thyronine (3-T(1)), in the serum of humans (n = 79) and wild Baikal seals (n = 37), by isotope ([(13)C(6)]-T(4))-dilution liquid chromatography (LC)-tandem mass spectrometry (MS/MS), and compared the TH levels with those measured by an electrochemiluminescent immunoassay (ECLIA) method. T(3) and T(4) were detected in all serum samples of both humans and Baikal seals, whereas T(1), 3,3'-T(2), and 3,5-T(2) were below the limit of detection (LOD). rT(3) was detected in Baikal seal sera at concentrations higher than T(3) in 28 seal samples, indicating an anomaly in deiodinase activity in Baikal seals. In humans, regression analyses of TH concentrations, measured by ECLIA and LC-MS/MS methods, showed significant correlations for T(4) (r = 0.852) and T(3) (r = 0.676; after removal of a serum sample with abnormal T(3) levels). In Baikal seals, a low correlation coefficient (r = 0.466) for T(4) levels and no correlation for T(3) levels (p = 0.093) were found between ECLIA and LC-MS/MS methods. These results suggest that interference by a nonspecific reaction against anti-T(3) and anti-T(4) antibodies used in the ECLIA can contribute to inaccuracies in TH measurement in Baikal seals. When the relationship between concentrations of THs in sera and dioxin-like toxic equivalents in blubber samples of Baikal seals (n = 19) was examined, a significantly negative correlation was found for serum T(4) levels measured by the LC-MS

  13. A sensitive LC-MS/MS method for measurement of organophosphorus pesticides and their oxygen analogs in air sampling matrices. (United States)

    Armstrong, Jenna L; Dills, Russell L; Yu, Jianbo; Yost, Michael G; Fenske, Richard A


    A rapid liquid chromatography tandem mass spectrometry (LC-MS/MS) method has been developed for determination of levels of the organophosphorus (OP) pesticides chlorpyrifos (CPF), azinphos methyl (AZM), and their oxygen analogs chlorpyrifos-oxon (CPF-O) and azinphos methyl-oxon (AZM-O) on common active air sampling matrices. XAD-2 resin and polyurethane foam (PUF) matrices were extracted with acetonitrile containing stable-isotope labeled internal standards (ISTD). Analysis was accomplished in Multiple Reaction Monitoring (MRM) mode, and analytes in unknown samples were identified by retention time (±0.1 min) and qualifier ratio (±30% absolute) as compared to the mean of calibrants. For all compounds, calibration linearity correlation coefficients were ≥0.996. Limits of detection (LOD) ranged from 0.15-1.1 ng/sample for CPF, CPF-O, AZM, and AZM-O on active sampling matrices. Spiked fortification recoveries were 78-113% from XAD-2 active air sampling tubes and 71-108% from PUF active air sampling tubes. Storage stability tests also yielded recoveries ranging from 74-94% after time periods ranging from 2-10 months. The results demonstrate that LC-MS/MS is a sensitive method for determining these compounds from two different matrices at the low concentrations that can result from spray drift and long range transport in non-target areas following agricultural applications. In an inter-laboratory comparison, the limit of quantification (LOQ) for LC-MS/MS was 100 times lower than a typical gas chromatography-mass spectrometry (GC-MS) method.

  14. Steroidal saponins from Yucca gloriosa L. rhizomes: LC-MS profiling, isolation and quantitative determination. (United States)

    Skhirtladze, Alexandre; Perrone, Angela; Montoro, Paola; Benidze, Mariam; Kemertelidze, Ether; Pizza, Cosimo; Piacente, Sonia


    The occurrence of steroidal saponins in the rhizomes of Yucca gloriosa has been detected by LC-MS. On the basis of the LC-MS analysis, five steroidal glycosides, including three spirostane, one furostane and one cholestane glycosides, along with seven known compounds have been isolated and characterized by ESI-MS and by the extensive use of 1D- and 2D-NMR experiments. Quantitative analysis of the steroidal glycosides in Y. gloriosa rhizomes was performed by an LC-MS method validated according to European Medicines Agency (EMEA) guidelines. The dried BuOH extract obtained from rhizomes contains more than 25% w/w of glycosides, thus Y. gloriosa rhizomes can be considered a rich source of steroidal glycosides. Copyright © 2010 Elsevier Ltd. All rights reserved.

  15. Application of LC/MS/MS Techniques to Development of US ... (United States)

    This presentation will describe the U.S. EPA’s drinking water and ambient water method development program in relation to the process employed and the typical challenges encountered in developing standardized LC/MS/MS methods for chemicals of emerging concern. The EPA’s Drinking Water Contaminant Candidate List and Unregulated Contaminant Monitoring Regulations, which are the driving forces behind drinking water method development, will be introduced. Three drinking water LC/MS/MS methods (Methods 537, 544 and a new method for nonylphenol) and two ambient water LC/MS/MS methods for cyanotoxins will be described that highlight some of the challenges encountered during development of these methods. This presentation will provide the audience with basic understanding of EPA's drinking water method development program and an introduction to two new ambient water EPA methods.

  16. Application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) in screening of high risk children with inherited metabolic diseases in northern China. (United States)

    Tu, Wenjun; Song, Xiaotao; Dai, Fang; Ho, James Jian


    To investigate the morbidity and distribution of 35 inherited metabolic diseases in high risk children by LC-MS/MS in northern China. The dry blood on filter papers, collected from 2760 children clinically suspected to have inherited metabolic diseases from more than sixty hospitals in north China, was tested by LC-MS/MS. The specimen was extracted out of the dry blood on filter paper, derivatized before being injected into LCMS/MS. The LC-MS/MS methodology used in the study was transferred from Pediatrix Medical Group (1301 Concord Terrace, Sunrise, FL 33323), validated in our lab and further compared with United States CDC standard. The positive results were further confirmed by gas chromatography-mass, other laboratory tests and clinical symptoms. 249 of the 2760 children (9%) were diagnosed with one or more of twenty-one disorders. Out of 249 patients, there are 41 (16.5%) fatty acid disorders, 71 (28.5%) amino acid diseases, and 137 (55%) organic acidemias. 48 of the 249 patients (19.3%) were neonates, including 11 (22.9%) with fatty acid disorders, 15 (31.3%) with amino acid diseases, and 22 (45.8%) with organic acidemias. 201 of the 249 patients were elder than 28 days, and was composed of 30 (14.9%) with fatty acid disorders, 56 (27.9%) with amino acid diseases, 115 (57.2%) with organic acidemias. The LC-MS/MS technology can be used to detect over 30 inherited metabolic disorders for Chinese pediatric clinic in a single collection of blood. The morbidity of IMD (9%) is relatively high among high risk children, thus we highly suggest that we shall provide initial screening of over 30 IMDs for the high risk children in China using the technology of LC-MS/MS.

  17. LC-MS-MS identification of drug metabolites obtained by metalloporphyrin mediated oxidation

    Directory of Open Access Journals (Sweden)

    Maurin Andrea J. M.


    Full Text Available In this paper we report the application of liquid chromatography-mass spectrometry (LC-MS-MS to the identification of the products formed by oxidation of albendazole and disopyramide with metalloporphyrins in dichloroethane, using iodosylbenzene as an oxygen donor. Our results show that LC-MS-MS is a powerful tool to study the in vitro metabolism of drugs, allowing the identification of known and unknown metabolites. In addition, it was observed that the catalyst system used resulted in the formation of the same metabolites as obtained in vivo, although for disopyramide other products were also observed.

  18. LC-MS-Based Metabolomic Investigation of Chemopreventive Phytochemical-Elicited Metabolic Events. (United States)

    Wang, Lei; Yao, Dan; Chen, Chi


    Phytochemicals are under intensive investigation for their potential use as chemopreventive agents in blocking or suppressing carcinogenesis. Metabolic interactions between phytochemical and biological system play an important role in determining the efficacy and toxicity of chemopreventive phytochemicals. However, complexities of phytochemical biotransformation and intermediary metabolism pose challenges for studying phytochemical-elicited metabolic events. Metabolomics has become a highly effective technical platform to detect subtle changes in a complex metabolic system. Here, using green tea polyphenols as an example, we describe a workflow of LC-MS-based metabolomics study, covering the procedures and techniques in sample collection, preparation, LC-MS analysis, data analysis, and interpretation.

  19. LC-MS/MS-based metabolites of Eurycoma longifolia (Tongkat Ali) in Malaysia (Perak and Pahang). (United States)

    Chua, Lee Suan; Amin, Nor Amaiza Mohd; Neo, Jason Chun Hong; Lee, Ting Hun; Lee, Chew Tin; Sarmidi, Mohamad Roji; Aziz, Ramlan Abdul


    A number of three LC-MS/MS hybrid systems (QTof, TripleTof and QTrap) has been used to profile small metabolites (m/z 100-1000) and to detect the targeted metabolites such as quassinoids, alkaloids, triterpene and biphenylneolignans from the aqueous extracts of Eurycoma longifolia. The metabolite profiles of small molecules showed four significant clusters in the principle component analysis for the aqueous extracts of E. longifolia, which had been collected from different geographical terrains (Perak and Pahang) and processed at different extraction temperatures (35°C and 100°C). A small peptide of leucine (m/z 679) and a new hydroxyl methyl β-carboline propionic acid have been identified to differentiate E. longifolia extracts that prepared at 35°C and 100°C, respectively. From the targeted metabolites identification, it was found that 3,4ɛ-dihydroeurycomanone (quassinoids) and eurylene (squalene-type triterpene) could only be detected in the Pahang extract, whereas canthin-6-one-3N-oxide could only be detected in the Perak extract. Overall, quassinoids were present in the highest concentration, particularly eurycomanone and its derivatives compared to the other groups of metabolites. However, the concentration of canthin-6-one and β-carboline alkaloids was significantly increased when the roots of the plant samples were extracted at 100°C. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Quantitative Determination of Ivermectin in Raw Milk Using Positive ESI LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Meenakshi Dahiya


    Full Text Available Ivermectin, a veterinary drug, is commonly used endectocide for animal husbandry. The drug is available in the form of subcutaneous or topical formulations. Its application may cause accumulation of its residues into the animal tissues, which ultimately find their way into the food products, such as milk and meat products. In order to determine the residues of ivermectin in milk, a comparatively simple, sensitive and rapid method was developed and validated using LC-MS/MS. The MRM transitions corresponding to m/z 892.71>569.6, 892.71>551.5 and 892.71>307.3 were used for the purpose of quantification and evaluation of other parameters of the method. The limit of detection of the method was found to be 0.1 μg/kg and the limit of quantitation was calculated as 0.2 μg/kg. The method was found to be linear in the range of 1.0 ng/mL to 100.0 ng/mL with correlation coefficient of 0.9992 for pure calibration curve and 0.9990 for the matrix- matched calibration curve. The recoveries of ivermectin from the spiked samples of raw milk were found between 85 to 105%.

  1. Simultaneous quantification of seven hippocampal neurotransmitters in depression mice by LC-MS/MS. (United States)

    Huang, Fei; Li, Jia; Shi, Hai-Lian; Wang, Ting-ting; Muhtar, Wahaf; Du, Min; Zhang, Bei-bei; Wu, Hui; Yang, Li; Hu, Zhi-bi; Wu, Xiao-jun


    There is no method available to simultaneously detect GABA, Glu, Epi, NE, DA, 5-HT and 5-HIAA in mouse hippocampus. A rapid and sensitive LC-MS/MS method has been developed for simultaneously measuring seven neurotransmitters in mouse hippocampus. The analytes were detected in positive mode with multiple reaction monitoring (MRM) and the procedure was completed in less than 9min. This method exhibited excellent linearity for all of the analytes with regression coefficients higher than 0.99, and showed good intra- and inter-day precisions (RSDneurotransmitters in a mouse depression model induced by successive methylprednisolone injections. The results indicated that this depression model was closely associated with the decreased level of Epi (p=0.002) and elevated ratio of 5-HIAA/5-HT (p=0.01), which has never been reported elsewhere. Compared with previous methods, current approach is more convenient without any pre-column derivatization of the analytes but enhances detectability with incremental neurotransmitter profile and shortens detection time. This work represents the first accurate simultaneous determination of seven neurotransmitters in the mouse depression model induced by methylprednisolone. The reliable method will benefit the research of neurological diseases with the altered neurotransmitter profile in brain. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Comparative proteomic analysis of Arthrobacter phenanthrenivorans Sphe3 on phenanthrene, phthalate and glucose. (United States)

    Vandera, Elpiniki; Samiotaki, Martina; Parapouli, Maria; Panayotou, George; Koukkou, Anna Irini


    In the present study, by applying comparative quantitative proteomics, we investigated the metabolic adaptation of Arthrobacter phenanthrenivorans Sphe3 when using phenanthrene, phthalate, glucose or glucose plus phenanthrene as sole carbon and energy sources. More than a third of the total Sphe3 proteins, with function prediction within the genome, were identified with confidence. Proteomic analysis data and annotated genomic information coincide, allowing us to clarify the phenanthrene catabolic pathway. We confirmed the implication of several proteins in aromatic substrate degradation by identifying those mediating the initial ring-hydroxylation and ring cleavage of phenanthrene to phthalate, phthalate degradation, as well as ortho- and meta-protocatechuate catabolism. Repression of catabolic genes by glucose was observed by both proteomic and transcriptional analyses. The presence of aromatic substrates resulted in changes in the abundance of proteins involved in substrate and amino acid metabolism, stress response, detoxification and membrane and cell wall metabolism. Uptake and transport associated proteins differ in the substrates used, indicating the use of different uptake mechanisms for transport of each compound in the Sphe3 cells. Our results also suggest the activation of a glyoxylate shunt in the presence of aromatic compounds, based on the up-regulation of the key enzymes of this pathway. A. phenanthrenivorans Sphe3, isolated from a creosote contaminated soil in Greece, can grow on phenanthrene as the sole source of carbon and energy. To explore the phenanthrene catabolic pathway by determining the key proteins involved in this pathway, as well as the global changes in proteins due to the adaptive response of Sphe3 cells grown on different substrates, we applied a gel-free quantitative proteomic analysis using nanoLC-MS/MS. To our knowledge this is the first study of comparative global proteomic changes occurring in the Sphe3 cells under exposure in

  3. Pharmacokinetic Evaluation of Empagliflozin in Healthy Egyptian Volunteers Using LC-MS/MS and Comparison with Other Ethnic Populations. (United States)

    Ayoub, Bassam M; Mowaka, Shereen; Elzanfaly, Eman S; Ashoush, Nermeen; Elmazar, Mohamed M; Mousa, Shaker A


    The present study considered the pharmacokinetic evaluation of empagliflozin after administration to Egyptian volunteers, and the results were compared with other ethnic populations. The FDA recognizes that standard methods of defining racial subgroups are necessary to compare results across pharmacokinetic studies and to assess potential subgroup differences. The design of the study was as an open labeled, randomized, one treatment, one period, single dose pharmacokinetic study. The main pharmacokinetic parameters estimated were C max , T max , t 1/2 , elimination rate constant, AUC 0-t and AUC 0-inf . The insignificant difference in pharmacokinetic parameters between Egyptians and white German subjects suggests that no dose adjustment should be considered with administration of 25 mg empagliflozin to Egyptian population. A new LC-MS/MS method was developed and validated, allowing sensitive estimation of empagliflozin (25-600 ng mL -1 ) in human plasma using dapagliflozin as an internal standard (IS). The method was applied successfully on the underlying pharmacokinetic study with enhanced sample preparation that involved liquid-liquid extraction. Multiple Reaction Monitoring (MRM) of the transition pairs of m/z 449.01 to 371.21 for empagliflozin and m/z 407.00 to 328.81 for dapagliflozin (IS) was employed utilizing negative mode Electro Spray Ionization (ESI). The validated LC-MS/MS method is suitable for further toxicodynamic and bioequivalence studies.

  4. Mini-Scale Isolation and Preparation of Plasma Membrane Proteins from Potato Roots for LC/MS Analysis. (United States)

    Jozefowicz, Anna M; Matros, Andrea; Witzel, Katja; Mock, Hans-Peter


    Plasma membrane (PM) proteins are of special interest due to their function in exchanging material and information with the external environment as well as their role in cellular regulation. In quantitative proteomic studies PM proteins are underrepresented mostly because they constitute only small percent of all membrane proteins. Strong demand is placed on plasma membrane enrichment methods. For decades two-phase partitioning Dextran T500/PEG 3350 isolation protocols were applied for many different animal and plant species and also a variety of tissue types. The typical quantity of material used in the enrichment protocols is 10-30 g of fresh weight. The main difficulty of working with in vitro cultivated plants is the low amount of material, especially when roots are examined. In addition, roots are frequently characterized by low protein concentrations. Our protocol established for roots of in vitro cultivated potato plants is adjusted to amounts of fresh weight not exceeding 7.5 g and allows studying the plasma membrane proteome by LC-MS.

  5. A Method for Microalgae Proteomics Analysis Based on Modified Filter-Aided Sample Preparation. (United States)

    Li, Song; Cao, Xupeng; Wang, Yan; Zhu, Zhen; Zhang, Haowei; Xue, Song; Tian, Jing


    With the fast development of microalgal biofuel researches, the proteomics studies of microalgae increased quickly. A filter-aided sample preparation (FASP) method is widely used proteomics sample preparation method since 2009. Here, a method of microalgae proteomics analysis based on modified filter-aided sample preparation (mFASP) was described to meet the characteristics of microalgae cells and eliminate the error caused by over-alkylation. Using Chlamydomonas reinhardtii as the model, the prepared sample was tested by standard LC-MS/MS and compared with the previous reports. The results showed mFASP is suitable for most of occasions of microalgae proteomics studies.

  6. Time-saving design of experiment protocol for optimization of LC-MS data processing in metabolomic approaches. (United States)

    Zheng, Hong; Clausen, Morten Rahr; Dalsgaard, Trine Kastrup; Mortensen, Grith; Bertram, Hanne Christine


    We describe a time-saving protocol for the processing of LC-MS-based metabolomics data by optimizing parameter settings in XCMS and threshold settings for removing noisy and low-intensity peaks using design of experiment (DoE) approaches including Plackett-Burman design (PBD) for screening and central composite design (CCD) for optimization. A reliability index, which is based on evaluation of the linear response to a dilution series, was used as a parameter for the assessment of data quality. After identifying the significant parameters in the XCMS software by PBD, CCD was applied to determine their values by maximizing the reliability and group indexes. Optimal settings by DoE resulted in improvements of 19.4% and 54.7% in the reliability index for a standard mixture and human urine, respectively, as compared with the default setting, and a total of 38 h was required to complete the optimization. Moreover, threshold settings were optimized by using CCD for further improvement. The approach combining optimal parameter setting and the threshold method improved the reliability index about 9.5 times for a standards mixture and 14.5 times for human urine data, which required a total of 41 h. Validation results also showed improvements in the reliability index of about 5-7 times even for urine samples from different subjects. It is concluded that the proposed methodology can be used as a time-saving approach for improving the processing of LC-MS-based metabolomics data.

  7. Simultaneous Determination of Multi-Mycotoxins in Cereal Grains Collected from South Korea by LC/MS/MS

    Directory of Open Access Journals (Sweden)

    Dong-Ho Kim


    Full Text Available An improved analytical method compared with conventional ones was developed for simultaneous determination of 13 mycotoxins (deoxynivalenol, nivalenol, 3-acetylnivalenol, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, T-2, HT-2, zearalenone, and ochratoxin A in cereal grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS after a single immunoaffinity column clean-up. The method showed a good linearity, sensitivity, specificity, and accuracy in mycotoxin determination by LC/MS/MS. The levels of 13 mycotoxins in 5 types of commercial grains (brown rice, maize, millet, sorghum, and mixed cereal from South Korea were determined in a total of 507 cereal grains. Mycotoxins produced from Fusarium sp. (fumonisins, deoxynivalenol, nivalenol, and zearalenone were more frequently (more than 5% and concurrently detected in all cereal grains along with higher mean levels (4.3–161.0 ng/g in positive samples than other toxins such as aflatoxins and ochratoxin A (less than 9% and below 5.2 ng/g in positive samples from other fungal species.

  8. MassCascade: Visual Programming for LC-MS Data Processing in Metabolomics. (United States)

    Beisken, Stephan; Earll, Mark; Portwood, David; Seymour, Mark; Steinbeck, Christoph


    Liquid chromatography coupled to mass spectrometry (LC-MS) is commonly applied to investigate the small molecule complement of organisms. Several software tools are typically joined in custom pipelines to semi-automatically process and analyse the resulting data. General workflow environments like the Konstanz Information Miner (KNIME) offer the potential of an all-in-one solution to process LC-MS data by allowing easy integration of different tools and scripts. We describe MassCascade and its workflow plug-in for processing LC-MS data. The Java library integrates frequently used algorithms in a modular fashion, thus enabling it to serve as back-end for graphical front-ends. The functions available in MassCascade have been encapsulated in a plug-in for the workflow environment KNIME, allowing combined use with e.g. statistical workflow nodes from other providers and making the tool intuitive to use without knowledge of programming. The design of the software guarantees a high level of modularity where processing functions can be quickly replaced or concatenated. MassCascade is an open-source library for LC-MS data processing in metabolomics. It embraces the concept of visual programming through its KNIME plug-in, simplifying the process of building complex workflows. The library was validated using open data.

  9. Quantification of free and total sialic acid excretion by LC-MS/MS

    NARCIS (Netherlands)

    van der Ham, Maria; Prinsen, Berthil H. C. M. T.; Huijmans, Jan G. M.; Abeling, Nicolaas G. G. M.; Dorland, Bert; Berger, Ruud; de Koning, Tom J.; de Sain-van der Velden, Monique G. M.


    The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC-MS/MS method for quantification of FSA and total sialic

  10. Comparison of MALDI-MSI and LC-MS for pharmacokinetic study of metformin

    Czech Academy of Sciences Publication Activity Database

    Strnad, Štěpán; Sýkora, D.; Cvačka, Josef; Maletínská, Lenka; Pirník, Z.; Majerčíková, Zuzana; Kuneš, Jaroslav; Vrkoslav, Vladimír


    Roč. 15, č. 1 (2017), s. 35 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : metformin * MALDI-MSI * LC-MS Subject RIV: CB - Analytical Chemistry, Separation

  11. Effect of trans fatty acid intake on LC-MS and NMR plasma profiles

    DEFF Research Database (Denmark)

    Gürdeniz, Gözde; Rago, Daniela; Bendsen, Nathalie Tommerup


    The consumption of high levels of industrial trans fatty acids (TFA) has been related to cardiovascular disease, diabetes and sudden cardiac death but the causal mechanisms are not well known. In this study, NMR and LC-MS untargeted metabolomics has been used as an approach to explore the impact...

  12. Sequential immunoaffinity-LC/MS assay for quantitation of a therapeutic protein in monkey plasma

    Directory of Open Access Journals (Sweden)

    Linzhi Chen


    Full Text Available Immunocapture-LC/MS has recently been used for quantitating therapeutic proteins/peptides and biomarkers in various matrices. The advantages of LC/MS quantitation include high speci­ficity and selectivity, wide dynamic range, and less susceptibility to interference from endogenous matrix components. We present a highly sensitive sequential immunoaffinity-LC/MS assay for quantitation of a biotherapeutic protein (39 kD in monkey plasma. The first immunocapture utilized a biotinylat­ed mouse anti-drug antibody to capture the drug in plasma. After tryptic digestion, a unique peptide from the drug was then captured by the sec­ond immunocapture using a mouse anti-peptide antibody for further sample purification. Samples analysis was performed on a microLC-triple quadrupole mass spectrometry system (MS/MS. Both immunocapture procedures were carried out in 96-well plates using a magnetic beads handler. The LLOQ of the assay is 50 pg/mL, which was approximately 100x more sensitive than a corresponding single immunocapture-LC/MS assay either using the anti-drug or anti-peptide antibody.

  13. LC-MS3 quantification of O-glycopeptides in human serum

    Czech Academy of Sciences Publication Activity Database

    Sanda, M.; Pompach, Petr; Benicky, J.; Goldman, R.


    Roč. 34, č. 16 (2013), s. 2342-2349 ISSN 0173-0835 R&D Projects: GA MŠk LH13051 Institutional support: RVO:61388971 Keywords : LC-MS3 * O-glycosylation * Quantification of glycopeptides Subject RIV: CE - Biochemistry Impact factor: 3.161, year: 2013

  14. Probabilistic model for untargeted peak detection in LC-MS using Bayesian statistics

    NARCIS (Netherlands)

    Woldegebriel, M.; Vivó-Truyols, G.


    We introduce a novel Bayesian probabilistic peak detection algorithm for liquid chromatography mass spectroscopy (LC-MS). The final probabilistic result allows the user to make a final decision about which points in a 2 chromatogram are affected by a chromatographic peak and which ones are only

  15. MassCascade: Visual Programming for LC-MS Data Processing in Metabolomics (United States)

    Beisken, Stephan; Earll, Mark; Portwood, David; Seymour, Mark; Steinbeck, Christoph


    Liquid chromatography coupled to mass spectrometry (LC-MS) is commonly applied to investigate the small molecule complement of organisms. Several software tools are typically joined in custom pipelines to semi-automatically process and analyse the resulting data. General workflow environments like the Konstanz Information Miner (KNIME) offer the potential of an all-in-one solution to process LC-MS data by allowing easy integration of different tools and scripts. We describe MassCascade and its workflow plug-in for processing LC-MS data. The Java library integrates frequently used algorithms in a modular fashion, thus enabling it to serve as back-end for graphical front-ends. The functions available in MassCascade have been encapsulated in a plug-in for the workflow environment KNIME, allowing combined use with e.g. statistical workflow nodes from other providers and making the tool intuitive to use without knowledge of programming. The design of the software guarantees a high level of modularity where processing functions can be quickly replaced or concatenated. MassCascade is an open-source library for LC-MS data processing in metabolomics. It embraces the concept of visual programming through its KNIME plug-in, simplifying the process of building complex workflows. The library was validated using open data. PMID:26279687

  16. Determination of Flavonoids and Resveratrol in Wine by Turbulent-Flow Chromatography-LC-MS

    NARCIS (Netherlands)

    Presta, M.A.; Bruyneel, B.; Zanella, R.; Kool, J.; Krabbe, J.G.; Lingeman, H.


    Turbulent-flow chromatography (TFC) on-line coupled to liquid chromatography mass spectrometry (LC-MS) is used to determine flavonoids and resveratrol in different types of wines. A fully automated system was developed in which 10 mL of sample (diluted wine) was passed over a TFC column, after which

  17. LC-MS analysis and effects of Malaysian propolis on insulin ...

    African Journals Online (AJOL)

    This study evaluated the phytochemical constituents using liquid chromatographmass spectrometry (LC-MS) analysis and in vivo effects of Malaysian propolis on plasma insulin, glucagon, oxidative stress status and histology of pancreas in diabetic rats. Analysis was carried out onwater (WEP) and ethanol (EEP) extracts of ...

  18. Characterization of bixin by LC-MS and LC-NMR. (United States)

    Rehbein, Jens; Dietrich, Benjamin; Grynbaum, Marc David; Hentschel, Petra; Holtin, Karsten; Kuehnle, Maximilian; Schuler, Paul; Bayer, Marc; Albert, Klaus


    An overview upon modern analytical techniques for the isolation, separation, and structural identification of the essential bioactive carotenoid bixin is given. Isolation from biological matrices is performed by matrix solid phase dispersion (MSPD). The extract is separated with shape-selective C(30 )columns. Structural assignment of the separated compounds is done by online LC-MS and capillary HPLC-NMR.

  19. Determination of selected bisphenols, parabens and estrogens in human plasma using LC-MS/MS

    Czech Academy of Sciences Publication Activity Database

    Kolatorova Sosvorova, L.; Chlupacova, T.; Vitku, J.; Vlk, Martin; Heráček, J.; Stárka, L.; Šaman, David; Šimková, M.; Hampl, R.


    Roč. 174, NOV 1 (2017), s. 21-28 ISSN 0039-9140 Institutional support: RVO:61389030 ; RVO:61388963 Keywords : Alternative bisphenol * Bisphenol A * Bisphenol F * Endocrine disruptor * lc-ms/ms * Paraben Subject RIV: CC - Organic Chemistry OBOR OECD: Analytical chemistry; Organic chemistry (UOCHB-X) Impact factor: 4.162, year: 2016

  20. Determination of chlormequat in pig serum and sow milk by LC-MS/MS

    DEFF Research Database (Denmark)

    Poulsen, Mette Erecius; Christensen, Hanne Bjerre; Sørensen, M.T.


    reproduction. Validation of the method was based on recovery tests at three spiking levels, determined as double determinations and repeated at least four times. Samples were extracted with methanol-water-acetic acid, centrifuged, filtrated and determined by LC-MS/MS. The mean recoveries were in the range 80...

  1. A comparative proteomics method for multiple samples based on a 18O-reference strategy and a quantitation and identification-decoupled strategy. (United States)

    Wang, Hongbin; Zhang, Yongqian; Gui, Shuqi; Zhang, Yong; Lu, Fuping; Deng, Yulin


    Comparisons across large numbers of samples are frequently necessary in quantitative proteomics. Many quantitative methods used in proteomics are based on stable isotope labeling, but most of these are only useful for comparing two samples. For up to eight samples, the iTRAQ labeling technique can be used. For greater numbers of samples, the label-free method has been used, but this method was criticized for low reproducibility and accuracy. An ingenious strategy has been introduced, comparing each sample against a 18 O-labeled reference sample that was created by pooling equal amounts of all samples. However, it is necessary to use proportion-known protein mixtures to investigate and evaluate this new strategy. Another problem for comparative proteomics of multiple samples is the poor coincidence and reproducibility in protein identification results across samples. In present study, a method combining 18 O-reference strategy and a quantitation and identification-decoupled strategy was investigated with proportion-known protein mixtures. The results obviously demonstrated that the 18 O-reference strategy had greater accuracy and reliability than other previously used comparison methods based on transferring comparison or label-free strategies. By the decoupling strategy, the quantification data acquired by LC-MS and the identification data acquired by LC-MS/MS are matched and correlated to identify differential expressed proteins, according to retention time and accurate mass. This strategy made protein identification possible for all samples using a single pooled sample, and therefore gave a good reproducibility in protein identification across multiple samples, and allowed for optimizing peptide identification separately so as to identify more proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Comparative proteomic analysis of pathogenic and non-pathogenic strains from the swine pathogen Mycoplasma hyopneumoniae

    Directory of Open Access Journals (Sweden)

    Klein Cátia S


    Full Text Available Abstract Background Mycoplasma hyopneumoniae is a highly infectious swine pathogen and is the causative agent of enzootic pneumonia (EP. Following the previous report of a proteomic survey of the pathogenic 7448 strain of swine pathogen, Mycoplasma hyopneumoniae, we performed comparative protein profiling of three M. hyopneumoniae strains, namely the non-pathogenic J strain and the two pathogenic strains 7448 and 7422. Results In 2DE comparisons, we were able to identify differences in expression levels for 67 proteins, including the overexpression of some cytoadherence-related proteins only in the pathogenic strains. 2DE immunoblot analyses allowed the identification of differential proteolytic cleavage patterns of the P97 adhesin in the three strains. For more comprehensive protein profiling, an LC-MS/MS strategy was used. Overall, 35% of the M. hyopneumoniae genome coding capacity was covered. Partially overlapping profiles of identified proteins were observed in the strains with 81 proteins identified only in one strain and 54 proteins identified in two strains. Abundance analysis of proteins detected in more than one strain demonstrates the relative overexpression of 64 proteins, including the P97 adhesin in the pathogenic strains. Conclusions Our results indicate the physiological differences between the non-pathogenic strain, with its non-infective proliferate lifestyle, and the pathogenic strains, with its constitutive expression of adhesins, which would render the bacterium competent for adhesion and infection prior to host contact.

  3. Glycomic profiling of tissue sections by LC-MS. (United States)

    Hu, Yunli; Zhou, Shiyue; Khalil, Sarah I; Renteria, Calvin L; Mechref, Yehia


    Because routine preparation of glycan samples involves multiple reaction and cleaning steps at which sample loss occurs, glycan analysis is typically performed using large tissue samples. This type of analysis yields no detailed molecular spatial information and requires special care to maintain proper storage and shipping conditions. We describe here a new glycan sample preparation protocol using minimized sample preparation steps and optimized procedures. Tissue sections and spotted samples first undergo on-surface enzymatic digestion to release N-glycans. The released glycans are then reduced and permethylated prior to online purification and LC-electrospray ionization (ESI)-MS analysis. The efficiency of this protocol was initially evaluated using model glycoproteins and human blood serum (HBS) spotted on glass or Teflon slides. The new protocol permitted the detection of permethylated N-glycans derived from 10 ng RNase B. On the other hand, 66 N-glycans were identified when injecting the equivalent of permethylated glycans derived from a 0.1-μL aliquot of HBS. On-tissue enzymatic digestion of nude mouse brain tissue permitted the detection of 43 N-glycans. The relative peak areas of these 43 glycans were comparable to those from a C57BL/6 mouse reported by the Consortium for Functional Glycomics (CFG). However, the sample size analyzed in the protocol described here was substantially smaller than for the routine method (submicrogram vs mg). The on-tissue N-glycan profiling method permits high sensitivity and reproducibility and can be widely applied to assess the spatial distribution of glycans associated with tissue sections, and may be correlated with immunoflourescence imaging when adjacent tissue sections are analyzed.

  4. Antibody-free LC-MS/MS quantification of rhTRAIL in human and mouse serum

    NARCIS (Netherlands)

    Wilffert, Daniel; Reis, C.R.; Hermans, Jos; Govorukhina, Natalia; Tomar, Tushar; de Jong, Steven; Quax, Wim J.; van de Merbel, Nico C.; Bischoff, Rainer


    The major challenge in targeted protein quantification by LC-MS/MS in serum lies in the complexity of the biological matrix with regard to the wide diversity of proteins and their extremely large dynamic concentration range. In this study, an LC-MS/MS method was developed for the simultaneous

  5. LC-MS/MS determination of pesticide residues in fruits and vegetables. (United States)

    Stachniuk, Anna; Szmagara, Agnieszka; Czeczko, Renata; Fornal, Emilia


    The aim of the research is to evaluate pesticide residue contamination of fresh and frozen fruits and vegetables, agricultural raw material, purchased from Polish farmers for production of frozen fruits and vegetables, and the estimation of the multiresidue method effectiveness expressed as the proportion of pesticides detected in food samples to the total number of pesticides analyzed by multiresidue methods. A total of 144 samples (of black currants, red currants, raspberries, cherries, strawberries, blackberries, cauliflowers and broccoli) were analyzed using LC-MS/MS method for the determination of 60 pesticides. QuEChERS extraction, matrix-matched calibration and dynamic multiple reaction monitoring method were used. Residues of 15 compounds, mainly fungicides and insecticides, were detected in 46 samples. The percentage of samples with residues above the maximum residue levels (MRL) was 15%, whereas samples with residues below MRL were 17%. A total of 13 samples contained more than one pesticide residue. Pesticide residues were detected most often in samples of black currants (50%), broccoli (36.4%), raspberries (29%) and red currants (21.8%). The most frequently detected pesticides were carbendazim and acetamiprid. The proportion of pesticides detected during our study to the total number of analyzed pesticides amounted to 25%. It was compared to literature findings. For three fourth of multiresidue methods, the proportion was below 50% for methods developed for the analysis of less than 100 pesticides, and below 30% for methods developed for the analysis of more than 100 pesticides. It appears that a lot of efforts and means is lost on pesticides never or rarely detected in examined samples. The workload and cost effectiveness of the development and application of multiresidue methods along with the range of pesticides covered by the method should be carefully and thoroughly considered anytime when a new method or workflow is developed. Including non

  6. Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development. (United States)

    Nie, Chun-Hong; Wan, Shi-Ming; Tomljanovic, Tea; Treer, Tomislav; Hsiao, Chung-Der; Wang, Wei-Min; Gao, Ze-Xia


    Intermuscular bones (IBs) and ribs both are a part of skeletal system in teleosts, but with different developing process. The chemical composition of fish IBs and ribs as well as the underlying mechanism about their development have not been investigated. In the present study, histological structures showed that one bone cavity containing osteoclasts were existed in ribs, but not in IBs of Megalobrama amblycephala. We constructed the first proteomics map for fish bones including IBs and ribs, and identified the differentially expressed proteins between IBs and ribs through iTRAQ LC-MS/MS proteomic analysis. The proteins extracted from IBs and ribs at 1- to 2-year old M. amblycephala were quantified 2,342 proteins, with 1,451 proteins annotated with GO annotation in biological processes, molecular function and cellular component. A number of bone related proteins as well as pathways were identified in the study. A total of 93 and 154 differently expressed proteins were identified in comparison groups of 1-IB-vs-1-Rib and 2-IB-vs-2-Rib, which indicated the obvious differences of chemical composition between these two bone tissues. The two proteins (vitronectin b precursor and matrix metalloproteinase-2) related to osteoclasts differentiation were significantly up-regulated in ribs compared with IBs (P development and differentiation. Subsequently, 11 and 13 candidate proteins in comparison group of 1-IB-vs-1-Rib and 1-IB-vs-2-IB related to bone development were validated by MRM assays. Our present study suggested the different key proteins involved in the composition of fish ribs and IBs as well as their growth development. These findings could provide important clues towards further understanding of fish skeletal system and the roles of proteins playing in regulating diverse biological processes in fish.

  7. Metabolic labeling and membrane fractionation for comparative proteomic analysis of Arabidopsis thaliana suspension cell cultures. (United States)

    Szymanski, Witold G; Kierszniowska, Sylwia; Schulze, Waltraud X


    Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% (1). Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient (2). Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures. We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K(15)NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest (3). By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or

  8. Comparative proteomics and codon substitution analysis reveal mechanisms of differential resistance to hypoxia in congeneric snails. (United States)

    Mu, Huawei; Sun, Jin; Cheung, Siu Gin; Fang, Ling; Zhou, Haiyun; Luan, Tiangang; Zhang, Huoming; Wong, Chris K C; Qiu, Jian-Wen


    Although high-throughput proteomics has been widely applied to study mechanisms of environmental adaptation, the conclusions from studies that are based on one species can be confounded by phylogeny. We compare the freshwater snail Pomacea canaliculata (a notorious invasive species) and its congener Pomacea diffusa (a non-invasive species) to understand the molecular mechanisms of their differential resistance to hypoxia. A 72-h acute exposure experiment showed that P. canaliculata is more tolerant to hypoxia than P. diffusa. The two species were then exposed to three levels of dissolved oxygen (6.7, 2.0 and 1.0mgL -1 ) for 8h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. The two species showed striking differences in protein expression profiles, with the more hypoxia tolerant P. canaliculata having more up-regulated proteins in signal transduction and down-regulated proteins in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis revealed five orthologous genes encoding differentially expressed proteins having clear signal of positive selection, indicating selection has acted on some of the hypoxia responsive genes. Our case study has highlighted the potential of integrated proteomics and comparative evolutionary analysis for understanding the genetic basis of adaptation to global environmental change in non-model species. Rapid globalization in recent decades has greatly facilitated species introduction around the world. Successfully established introduced species, so-called invasive species, have threatened the invaded ecosystems. There has been substantial interest in studying how invasive species respond to extreme environmental conditions because the results can help not only predict their range of expansion and manage their impact, but also may reveal the adaptive mechanisms underlying their invasiveness. Our study has adopted a comparative approach to study the differential physiological and proteomic responses of two

  9. Comparative proteomics and codon substitution analysis reveal mechanisms of differential resistance to hypoxia in congeneric snails

    KAUST Repository

    Mu, Huawei


    Although high-throughput proteomics has been widely applied to study mechanisms of environmental adaptation, the conclusions from studies that are based on one species can be confounded by phylogeny. We compare the freshwater snail Pomacea canaliculata (a notorious invasive species) and its congener Pomacea diffusa (a non-invasive species) to understand the molecular mechanisms of their differential resistance to hypoxia. A 72-h acute exposure experiment showed that P. canaliculata is more tolerant to hypoxia than P. diffusa. The two species were then exposed to three levels of dissolved oxygen (6.7, 2.0 and 1.0mgL−1) for 8h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. The two species showed striking differences in protein expression profiles, with the more hypoxia tolerant P. canaliculata having more up-regulated proteins in signal transduction and down-regulated proteins in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis revealed five orthologous genes encoding differentially expressed proteins having clear signal of positive selection, indicating selection has acted on some of the hypoxia responsive genes. Our case study has highlighted the potential of integrated proteomics and comparative evolutionary analysis for understanding the genetic basis of adaptation to global environmental change in non-model species. SignificanceRapid globalization in recent decades has greatly facilitated species introduction around the world. Successfully established introduced species, so-called invasive species, have threatened the invaded ecosystems. There has been substantial interest in studying how invasive species respond to extreme environmental conditions because the results can help not only predict their range of expansion and manage their impact, but also may reveal the adaptive mechanisms underlying their invasiveness. Our study has adopted a comparative approach to study the differential physiological and proteomic

  10. Rapid Determination of Isomeric Benzoylpaeoniflorin and Benzoylalbiflorin in Rat Plasma by LC-MS/MS Method

    Directory of Open Access Journals (Sweden)

    Chuanqi Zhou


    Full Text Available Benzoylpaeoniflorin (BP is a potential therapeutic agent against oxidative stress related Alzheimer’s disease. In this study, a more rapid, selective, and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS method was developed to determine BP in rat plasma distinguishing with a monoterpene isomer, benzoylalbiflorin (BA. The method showed a linear response from 1 to 1000 ng/mL (r>0.9950. The precision of the interday and intraday ranged from 2.03 to 12.48% and the accuracy values ranged from −8.00 to 10.33%. Each running of the method could be finished in 4 minutes. The LC-MS/MS method was validated for specificity, linearity, precision, accuracy, recovery, and stability and was found to be acceptable for bioanalytical application. Finally, this fully validated method was successfully applied to a pharmacokinetic study in rats following oral administration.

  11. How much separation for LC-MS/MS quantitative bioanalysis of drugs and metabolites? (United States)

    Tan, Aimin; Fanaras, John C


    LC-MS/MS has been the dominant analytical technology for quantitative bioanalysis of drugs and metabolites for more than two decades. Despite this, a very fundamental question like how much separation is required for LC-MS/MS quantitative bioanalysis of drugs and metabolites has not been adequately addressed. Some think that no or only very limited separation is necessary thanks to the unparalleled selectivity offered by tandem mass spectrometry. Others think that the more separation, the better, because of the potential detrimental impact of matrix effect (ion suppression or enhancement). Still others just use a rule-of-thumb approach by keeping the adjusted retention/capacity factor always between 2 and 5. The purpose of this article is to address this fundamental question through rational thinking together with various real case examples drawn from regulated bioanalytical laboratories. Copyright © 2018 Elsevier B.V. All rights reserved.

  12. Development of an LC/MS method for the trace analysis of triacetone triperoxide (TATP). (United States)

    Widmer, Leo; Watson, Stuart; Schlatter, Konrad; Crowson, Andrew


    The detection and quantification of triacetone triperoxide (TATP) using LC/MS is investigated. GC/MS analysis of TATP is hindered by stationary phase activation in very short periods of time. Due to the lower temperatures used in LC. this problem is not encountered. This study presents a method that is suitable for the detection of TATP at levels as low as 100 pg microl(-1) (10 ng per 100 microl). Initial findings are also reported for the investigation of a secondary chromatographic peak, which is thought to be caused by separation of two conformers. This study concludes that LC/MS is a suitable technique for the analysis of trace levels of TATP.

  13. Improved Label-Free LC-MS Analysis by Wavelet-Based Noise Rejection

    Directory of Open Access Journals (Sweden)

    Salvatore Cappadona


    Full Text Available Label-free LC-MS analysis allows determining the differential expression level of proteins in multiple samples, without the use of stable isotopes. This technique is based on the direct comparison of multiple runs, obtained by continuous detection in MS mode. Only differentially expressed peptides are selected for further fragmentation, thus avoiding the bias toward abundant peptides typical of data-dependent tandem MS. The computational framework includes detection, alignment, normalization and matching of peaks across multiple sets, and several software packages are available to address these processing steps. Yet, more care should be taken to improve the quality of the LC-MS maps entering the pipeline, as this parameter severely affects the results of all downstream analyses. In this paper we show how the inclusion of a preprocessing step of background subtraction in a common laboratory pipeline can lead to an enhanced inclusion list of peptides selected for fragmentation and consequently to better protein identification.

  14. A survey of free glutamic acid in foods using a robust LC-MS/MS method. (United States)

    Cebi, Nur; Dogan, Canan Ekinci; Olgun, Elmas Oktem; Sagdic, Osman


    An effective and simultaneous liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used with the aim of quantifying monosodium glutamate (MSG) in foodstuffs, such as chips, taste cubes, sauces and soups. The results were linear (R 2  = 1), with very low LOD and LOQ values, 1.0 µg/kg, 5.0 µg/kg, respectively. Excellent repeatability and reproducibility were also achieved. This highly sensitive and robust LC-MS/MS technique was applied successfully for the detection and quantification of MSG in a wide variety of foodstuffs. MSG contents ranged from 0.01 g/100 g to 15.39 g/100 g in food samples. Importantly, determination of free glutamic acid in the daily diet could also prevent various side effects associated with consumption of excess free glutamic acid. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. A Modified LC/MS/MS Method with Enhanced Sensitivity for the Determination of Scopolamine in Human Plasma (United States)

    Wang, Zuwei; Vaksman, Zalman; Putcha, Lakshmi


    Intranasal scopolamine is a choice drug for the treatment of motion sickness during space flight because of its quick onset of action, short half-life and favorable sideeffects profile. The dose administered usually ranges between 0.1 and 0.4 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids using existing sensitive LC/MS/MS method, especially when the biological sample volumes are limited. To measure scopolamine in human plasma to facilitate pharmacokinetic evaluation of the drug, we developed a sensitive LC/MS/MS method using 96 well micro elution plates for solid phase extraction (SPE) of scopolamine in human plasma. Human plasma (100-250 micro L) were loaded onto Waters Oasis HLB 96 well micro elution plate and eluted with 50 L of organic solvent without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 3 minutes. The mobile phase for separation was 80:20 (v/v) methanol: ammonium acetate (30 mM) in water. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 right arrow 138.1 and internal standard hyoscyamine m/z = 290.2 right arrow 124.1. The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at about 1.1 and 1.7 min respectively. The linear range is 25-10000 pg/mL for scopolamine in human plasma with correlation coefficients greater than 0.99 and CV less than 0.5%. The intra-day and inter-day CVs are less than 15% for quality control samples with concentrations of 75,300, and 750 pg/mL of scopolamine in human plasma. SPE using 96 well micro elution plates allows rapid sample preparation and enhanced sensitivity for the LC/MS

  16. The utility of qNMR to improve accuracy and precision of LC-MS bioanalysis


    Dieter M. Drexler; Colleen A. McNaney; Yingzi Wang; Xiaohua Huang; Michael D. Reily


    In LC-MS bioanalysis, samples and analytes are quantified against calibration solutions and curves which are derived via serial dilution from stock solutions that are prepared from dry reference standards. The analytical errors associated with the mass and volume measurements required for this preparation of stock solutions can culminate in a variance which might affect bioanalytical data accuracy. Especially in the case of extended studies with intermittent sample analysis, the multiple prep...

  17. Evaluation of Tamoxifen and metabolites by LC-MS/MS and HPLC Methods


    Heath, D.D.; Flatt, S.W.; Wu, A.H.B.; Pruitt, M.A.; Rock, C.L.


    Epidemiological and laboratory evidence suggests that quantification of serum or plasma levels of tamoxifen and the metabolites of tamoxifen, 4-hydroxy-N-desmethyl-tamoxifen (endoxifen), Z-4-hydroxy-tamoxifen (4HT), N-desmethyl-tamoxifen (ND-tam) is a clinically useful tool in the assessment and monitoring of breast cancer status in patients taking adjuvant tamoxifen. A liquid chromatographic mass spectrometric method (LC-MS/MS) was used to measure the blood levels of tamoxifen and the metabo...

  18. Quantifying MMA by SLE LC-MS/MS: Unexpected challenges in assay development. (United States)

    Lo, Sheng-Ying; Gordon, Cindy; Sadilkova, Katerina; Jack, Rhona M; Dickerson, Jane A


    Analysis of serum/plasma methylmalonic acid (MMA) is important for the diagnosis and management of methylmalonic acidemia in pediatric populations. This work focuses on developing and validating a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor methylmalonic acidemia using a simple method preparation. MMA and stable isotope labeled d3-MMA was extracted using supported liquid extraction (SLE). Assay imprecision, bias, linearity, recovery and carryover were determined. The relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) was also evaluated using historical paired results from 51 unique individuals. Baseline separation between MMA and succinic acid was completed in 7min. The assay was linear from 0.1 to 500μM. The intra-day and inter-day imprecision CV ranged from 4.1 to 13.2% (0.3 to 526μM) and 5.0 to 15.7% (0.3 to 233μM), respectively. Recovery ranged from 93 to 125%. The correlation with a national reference laboratory LC-MS/MS assay showed a Deming regression of 1.026 and intercept of -1.335. Carryover was determined to be MMA and C3-acylcarnitine. This report describes the first LC-MS/MS method using SLE for MMA extraction. In addition, we illustrate the challenges encountered during this method development that should be assessed and resolved by any laboratory implementing a SLE LC-MS/MS assay designed to quantify analytes across several orders of magnitude. Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  19. Specific determination of clinical and toxicological important substances in biological samples by LC-MS

    International Nuclear Information System (INIS)

    Mitulovic, G.


    This thesis of this dissertation is the specific determination of clinical and toxicological important substances in biological samples by LC-MS. Nicotine was determined in serum after application of nicotine plaster and nicotine nasal spray with HPLC-ESI-MS. Cotinine was determined direct in urine with HPLC-ESI-MS. Short time anesthetics were determined in blood and cytostatics were determined in liquor with HPLC-ESI-MS. (botek)

  20. New method for quantification of gasotransmitter hydrogen sulfide in biological matrices by LC-MS/MS


    Bo Tan; Sheng Jin; Jiping Sun; Zhongkai Gu; Xiaotian Sun; Yichun Zhu; Keke Huo; Zonglian Cao; Ping Yang; Xiaoming Xin; Xinhua Liu; Lilong Pan; Furong Qiu; Jian Jiang; Yiqun Jia


    Hydrogen sulfide exists widely in mammalian tissues and plays a vital role in physiological and pathophysiological processes. However, striking differences with orders of magnitude were observed for the detected hydrogen sulfide concentrations in biological matrices among different measurements in literature, which lead to the uncertainty for examination the biological relevance of hydrogen sulfide. Here, we developed and validated a liquid chromatography- mass spectrometry (LC-MS/MS) method ...

  1. An improved method for fast and selective separation of carotenoids by LC-MS. (United States)

    Abate-Pella, Daniel; Freund, Dana M; Slovin, Janet P; Hegeman, Adrian D; Cohen, Jerry D


    Carotenoids are a large class of compounds that are biosynthesized by condensation of isoprene units in plants, fungi, bacteria, and some animals. They are characteristically highly conjugated through double bonds, which lead to many isomers as well susceptibility to oxidation and other chemical modifications. Carotenoids are important because of their potent antioxidant activity and are the pigments responsible for color in a wide variety of foods. Human consumption is correlated to many health benefits including prevention of cancer, cardiovascular disease, and age-related disease. Extreme hydrophobicity, poor stability, and low concentration in biological samples make these compounds difficult to analyze and difficult to develop analytical methods for aimed towards identification and quantification. Examples in the literature frequently report the use of exotic stationary phases, solvents, and additives, such as ethyl acetate, dichloromethane, and methyl tert-butyl ether that are incompatible with liquid chromatography mass spectrometry (LC-MS). In order to address these issues, we implemented the use of LC-MS friendly conditions using a low-hydrophobicity cyano-propyl column (Agilent Zorbax SB-CN). We successfully differentiated between isomeric carotenoids by optimizing two gradient methods and using a mixture of 11 standards and LC-MS in positive ionization mode. Three complex biological samples from strawberry leaf, chicken feed supplement, and the photosynthetic bacterium Chloroflexus aurantiacus were analyzed and several carotenoids were resolved in these diverse backgrounds. Our results show this methodology is a significant improvement over other alternatives for analyzing carotenoids because of its ease of use, rapid analysis time, high selectivity, and, most importantly, its compatibility with typical LC-MS conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for analysis of endogenous steroids in the luteal phase and early pregnancy in dogs: a pilot study. (United States)

    Holst, Bodil S; Kushnir, Mark M; Bergquist, Jonas


    Blood samples from dogs are often limited in volume, only allowing few steroids to be quantified with immunoassays. In addition, immunoassays may be compromised by interferences such as anti-reagent antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be used for the simultaneous quantitation of several steroids. This has not been described in dogs before. The aims were to use LC-MS/MS to study steroid profiles in early pregnancy and luteal phase in dogs, and to determine if differences exist between pregnant (P) and nonpregnant (NP) dogs. Nine female dogs were included, 4 during a NP luteal phase, 4 during a P luteal phase, and one during one NP and one P luteal phase. Blood samples were collected around the time of the LH surge (Day 0) and on Day 26. Serum was analyzed for 5 classes of steroids, including glucocorticoids, androgens, estrogens, pregnanes, and progestins, using LC-MS/MS methods. The concentration of progesterone was significantly higher on Day 26 in P than in NP bitches. Distribution of concentrations of glucocorticoids, androgens, estrogens, or pregnanes in P and NP dogs were not statistically different. The predominating glucocorticoid was cortisol, and dihydroepiandrosterone (DHEA) was the predominating androgen. Concentration of estrone was comparable to oestradiol, whereas concentrations of pregnenolone were higher than those of 17-OH pregnenolone. Only concentration of progesterone differed between P and NP bitches, being significantly higher on Day 26 in P than in NP bitches. LC-MS/MS offers interesting possibilities for studies of canine reproductive endocrinology. © 2015 American Society for Veterinary Clinical Pathology.

  3. Ex vivo investigation of ocular tissue distribution following intravitreal administration of connexin43 mimetic peptide using the microdialysis technique and LC-MS/MS. (United States)

    Bisht, Rohit; Mandal, Abhirup; Rupenthal, Ilva D; Mitra, Ashim K


    This study aimed to develop and evaluate an ex vivo eye model for intravitreal drug sampling and tissue distribution of connexin43 mimetic peptide (Cx43MP) following intravitreal injection using the microdialysis technique and LC-MS/MS. An LC-MS/MS method was developed, validated, and applied for quantification of Cx43MP in ocular tissues. Microdialysis probes were calibrated for in vitro recovery studies. Bovine eyes were fixed in a customized eye holder and after intravitreal injection of Cx43MP, microdialysis probes were implanted in the vitreous body. Vitreous samples were collected at particular time intervals over 24 h. Moreover, 24 and 48 h after intravitreal injection ocular tissues were collected, processed, and analyzed for Cx43MP concentrations using LC-MS/MS. The LC-MS/MS method showed good linearity (r 2  = 0.9991). The mean percent recovery for lower (LQC), medium (MQC), and higher quality control (HQC) (0.244, 3.906, and 125 μg/mL) was found to be 83.83, 84.92, and 94.52, respectively, with accuracy ranges between 96 and 99 % and limits of detection (LOD) and quantification (LOQ) of 0.122 and 0.412 μg/mL. The in vitro recovery of the probes was found to be over 80 %. As per microdialysis sample analysis, the Cx43MP concentration was found to increase slowly in the vitreous body up to 16 h and thereafter declined. After 48 h, the Cx43MP concentration was higher in vitreous, cornea, and retina compared to lens, iris, and aqueous humor. This ex vivo model may therefore be a useful tool to investigate intravitreal kinetics and ocular disposition of therapeutic molecules after intravitreal injection.

  4. Relationship between plasma and salivary melatonin and cortisol investigated by LC-MS/MS. (United States)

    van Faassen, Martijn; Bischoff, Rainer; Kema, Ido P


    Disturbance of the circadian rhythm has been associated with disease states, such as metabolic disorders, depression and cancer. Quantification of the circadian markers such as melatonin and cortisol critically depend on reliable and reproducible analytical methods. Previously, melatonin and cortisol were primarily analyzed separately, mainly using immunoassays. Here we describe the validation and application of a high-throughput liquid chromatography in combination with mass spectrometry (LC-MS/MS) method for the combined analysis of melatonin and cortisol in plasma and saliva. The LC-MS/MS method was validated according to international validation guidelines. We used this method to analyze total plasma, free plasma (as obtained by equilibrium dialysis) and saliva melatonin and cortisol in healthy adults. Validation results for plasma and saliva melatonin and cortisol were well within the international validation criteria. We observed no difference between saliva collected by passive drooling or Salivette. Moreover, we noted a significant difference in saliva vs. free plasma melatonin. We observed on average 36% (95% CI: 4%-60%) higher salivary melatonin levels in comparison to free plasma melatonin, suggestive of local production of melatonin in the salivary glands. The novel outcome of this study is probably due to the high precision of our LC-MS/MS assay. These outcomes illustrate the added value of accurate and sensitive mass spectrometry based methods for the quantification of neuroendocrine biomarkers.

  5. Study of forced degradation behavior of pramlintide acetate by HPLC and LC-MS. (United States)

    Yuan, Yu; Li, Yuan-Bo; Tai, Zheng-Fu; Xie, Yi-Peng; Pu, Xu-Feng; Gao, Jian


    Pramlintide acetate (Symlin ® ), a synthetic analogue of the human hormone amylin. It was approved in March 2005 as a subcutaneous injection for the adjunctive treatment of patients who have type 1 or 2 diabetes mellitus. The objective of current investigation was to study the degradation behavior of pramlintide acetate under different ICH recommended stress conditions by HPLC and LC-MS. Pramlintide acetate was subjected to stress conditions of hydrolysis (acidic or alkaline), oxidation, photolysis and thermal decomposition. Extensive degradation products were observed under the hydrolysis, oxidation or thermal stress conditions, while minimal degradation was found in the photolytic conditions. Successful separation of drug from the degradation products was achieved by the validated chromatography (RP-HPLC and SCX-HPLC) methods. Subsequent to isolation, the molecular weight of each component was determined by LC-MS. The LC-MS m/z values and fragmentation patterns of 4 impurities matched with the predicted degradation products of pramlintide acetate. Copyright © 2017. Published by Elsevier B.V.

  6. Analysis of catecholamines in urine by unique LC/MS suitable ion-pairing chromatography. (United States)

    Bergmann, Marianne L; Sadjadi, Seyed; Schmedes, Anne


    The catecholamines, epinephrine (E) and norepinephrine (NE) are small polar, hydrophilic molecules, posing significant challenges to liquid chromatography - tandem mass spectrometry (LC-MS/MS) method development. Specifically, these compounds show little retention on conventional reversed-phase liquid chromatography columns. This work presents development and validation of an LC-MS/MS method for determining catecholamines in urine, based on a new approach to ion-pairing chromatography (IPC), in which the ion-pairing reagent (IPR), 1-Heptane Sulfonic Acid (HSA), is added to the extracted samples instead of the mobile phases. A Hamilton STARlet workstation carried out the solid phase extraction of urine samples. The extracted samples were diluted with 60mmol/L HSA and injected on a Kinetex core-shell biphenyl column with conventional LC-MS/MS suitable mobile phases. Chromatographic separation of E and NE was achieved successfully with very stable retention times (RT). In 484 injections, the RTs were steady with a CV of less than ±4%. Furthermore, HSA was separated from E and NE, allowing HSA to be diverted to waste instead of entering the mass spectrometer ion chamber. The method was validated with good analytical performance, and even though the analysis for urinary catecholamines is increasingly being replaced by plasma free metanephrines in diagnosing pheochromocytomas, this work represents the application of a new analytical technique that can be transferred to other small polar molecules, that are difficult to chromatograph on traditional reversed phase columns. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. 2-Hydrazinoquinoline as a Derivatization Agent for LC-MS-Based Metabolomic Investigation of Diabetic Ketoacidosis (United States)

    Lu, Yuwei; Yao, Dan; Chen, Chi


    Short-chain carboxylic acids, aldehydes and ketones are products and regulators of many important metabolic pathways. Their levels in biofluids and tissues reflect the status of specific metabolic reactions, the homeostasis of the whole metabolic system and the wellbeing of a biological entity. In this study, the use of 2-hydrazinoquinoline (HQ) as a novel derivatization agent was explored and optimized for simultaneous liquid chromatography-mass spectrometry (LC-MS) analysis of carboxylic acids, aldehydes and ketones in biological samples. The formation of carboxylic acid derivative is attributed to the esterification reaction between HQ and a carboxyl group, while the production of aldehyde and ketone derivatives is through the formation of Schiff bases between HQ and a carbonyl group. The compatibility of HQ with biological samples was demonstrated by derivatizing urine, serum and liver extract samples. Using this HQ-based approach, the kinetics of type 1 diabetes-induced metabolic changes was characterized by the LC-MS-based metabolomic analysis of urine samples from streptozotocin (STZ)-treated mice. Subsequently, carboxylic acid, aldehyde and ketone metabolites associated with STZ-elicited disruption of nutrient and energy metabolism were conveniently identified and elucidated. Overall, HQ derivatization of carboxylic acids, aldehydes and ketones could serve as a useful tool for the LC-MS-based metabolomic investigation of endogenous metabolism. PMID:24958262

  8. Proteomic Signatures of Thymomas (United States)

    Shilo, Konstantin; Hitchcock, Charles L.; Freitas, Michael A.


    Based on the histological features and outcome, the current WHO classification separates thymomas into A, AB, B1, B2 and B3 subtypes. It is hypothesized that the type A thymomas are derived from the thymic medulla while the type B thymomas are derived from the cortex. Due to occasional histological overlap between the tumor subtypes creating difficulties in their separation, the aim of this study was to provide their proteomic characterization and identify potential immunohistochemical markers aiding in tissue diagnosis. Pair-wise comparison of neoplastic and normal thymus by liquid chromatography tandem mass spectrometry (LC-MS/MS) of formalin fixed paraffin embedded tissue revealed 61 proteins differentially expressed in thymomas compared to normal tissue. Hierarchical clustering showed distinct segregation of subtypes AB, B1 and B2 from that of A and B3. Most notably, desmoyokin, a protein that is encoded by the AHNAK gene, was associated with type A thymomas and medulla of normal thymus, by LC-MS/MS and immunohistochemistry. In this global proteomic characterization of the thymoma, several proteins unique to different thymic compartments and thymoma subtypes were identified. Among differentially expressed proteins, desmoyokin is a marker specific for thymic medulla and is potentially promising immunohistochemical marker in separation of type A and B3 thymomas. PMID:27832160

  9. Proteomic Signatures of Thymomas.

    Directory of Open Access Journals (Sweden)

    Linan Wang

    Full Text Available Based on the histological features and outcome, the current WHO classification separates thymomas into A, AB, B1, B2 and B3 subtypes. It is hypothesized that the type A thymomas are derived from the thymic medulla while the type B thymomas are derived from the cortex. Due to occasional histological overlap between the tumor subtypes creating difficulties in their separation, the aim of this study was to provide their proteomic characterization and identify potential immunohistochemical markers aiding in tissue diagnosis. Pair-wise comparison of neoplastic and normal thymus by liquid chromatography tandem mass spectrometry (LC-MS/MS of formalin fixed paraffin embedded tissue revealed 61 proteins differentially expressed in thymomas compared to normal tissue. Hierarchical clustering showed distinct segregation of subtypes AB, B1 and B2 from that of A and B3. Most notably, desmoyokin, a protein that is encoded by the AHNAK gene, was associated with type A thymomas and medulla of normal thymus, by LC-MS/MS and immunohistochemistry. In this global proteomic characterization of the thymoma, several proteins unique to different thymic compartments and thymoma subtypes were identified. Among differentially expressed proteins, desmoyokin is a marker specific for thymic medulla and is potentially promising immunohistochemical marker in separation of type A and B3 thymomas.

  10. Informed-Proteomics: open-source software package for top-down proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jungkap; Piehowski, Paul D.; Wilkins, Christopher; Zhou, Mowei; Mendoza, Joshua; Fujimoto, Grant M.; Gibbons, Bryson C.; Shaw, Jared B.; Shen, Yufeng; Shukla, Anil K.; Moore, Ronald J.; Liu, Tao; Petyuk, Vladislav A.; Tolić, Nikola; Paša-Tolić, Ljiljana; Smith, Richard D.; Payne, Samuel H.; Kim, Sangtae


    Top-down proteomics involves the analysis of intact proteins. This approach is very attractive as it allows for analyzing proteins in their endogenous form without proteolysis, preserving valuable information about post-translation modifications, isoforms, proteolytic processing or their combinations collectively called proteoforms. Moreover, the quality of the top-down LC-MS/MS datasets is rapidly increasing due to advances in the liquid chromatography and mass spectrometry instrumentation and sample processing protocols. However, the top-down mass spectra are substantially more complex compare to the more conventional bottom-up data. To take full advantage of the increasing quality of the top-down LC-MS/MS datasets there is an urgent need to develop algorithms and software tools for confident proteoform identification and quantification. In this study we present a new open source software suite for top-down proteomics analysis consisting of an LC-MS feature finding algorithm, a database search algorithm, and an interactive results viewer. The presented tool along with several other popular tools were evaluated using human-in-mouse xenograft luminal and basal breast tumor samples that are known to have significant differences in protein abundance based on bottom-up analysis.

  11. Analytical and clinical performance of the new Fujirebio 25-OH vitamin D assay, a comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and three other automated assays


    Saleh, Lanja; Mueller, Daniel; von Eckardstein, Arnold


    BACKGROUND: We evaluated the analytical and clinical performance of the new Lumipulse® G 25-OH vitamin D assay from Fujirebio, and compared it to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and three other commercial automated assays. METHODS: Total 25 hydroxy vitamin D (25(OH)D) levels were measured in 100 selected serum samples from our routine analysis with Fujirebio 25(OH)D assay. The results were compared with those obtained with LC-MS/MS and three other automat...

  12. Quantitative performance of a quadrupole-orbitrap-MS in targeted LC-MS determinations of small molecules. (United States)

    Grund, Baptiste; Marvin, Laure; Rochat, Bertrand


    High-resolution mass spectrometry (HRMS) has been associated with qualitative and research analysis and QQQ-MS with quantitative and routine analysis. This view is now challenged and for this reason, we have evaluated the quantitative LC-MS performance of a new high-resolution mass spectrometer (HRMS), a Q-orbitrap-MS, and compared the results obtained with a recent triple-quadrupole MS (QQQ-MS). High-resolution full-scan (HR-FS) and MS/MS acquisitions have been tested with real plasma extracts or pure standards. Limits of detection, dynamic range, mass accuracy and false positive or false negative detections have been determined or investigated with protease inhibitors, tyrosine kinase inhibitors, steroids and metanephrines. Our quantitative results show that today's available HRMS are reliable and sensitive quantitative instruments and comparable to QQQ-MS quantitative performance. Taking into account their versatility, user-friendliness and robustness, we believe that HRMS should be seen more and more as key instruments in quantitative LC-MS analyses. In this scenario, most targeted LC-HRMS analyses should be performed by HR-FS recording virtually "all" ions. In addition to absolute quantifications, HR-FS will allow the relative quantifications of hundreds of metabolites in plasma revealing individual's metabolome and exposome. This phenotyping of known metabolites should promote HRMS in clinical environment. A few other LC-HRMS analyses should be performed in single-ion-monitoring or MS/MS mode when increased sensitivity and/or detection selectivity will be necessary. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Vitamins D and A can be successfully measured by LC-MS/MS in cord blood diluted plasma. (United States)

    Albarhani, Ali A; Collier, Fiona; Greaves, Ronda F; Ponsonby, Anne-Louise; Allen, Katrina J; Vuillermin, Peter J; Roche, Peter; Clarke, Michael W


    In widely used protocols for the collection and isolation of cord blood mononuclear cells, investigators are left with substantial volumes of diluted plasma which could be used for other measurements. The aim of this study was to ascertain the validity of umbilical cord blood (UCB) diluted plasma samples for vitamin D, A and E analysis compared to UCB serum samples. Twenty UCB matched samples of diluted plasma and serum were collected. The samples were analysed by two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods on two separate occasions. The results of 25(OH)D3 obtained by the two laboratories demonstrated close agreement with a mean difference of 0.14nmol/L [95% confidence interval (95% CI), -6.8 to 7.1]. Both methods demonstrate close agreement for 25(OH)D3 in UCB serum versus diluted UCB plasma; mean difference 2.2nmol/L [95% CI, -9.5 to 13.9] and 4.1nmol/L [95% CI, -14.5 to 6.1] for the results from Lab A and Lab B, respectively. Vitamin A was quantified by Lab A in UCB serum and diluted UCB plasma; mean difference 0.07μmol/L [95% CI, -0.41 to 0.28]. Results of 25(OH)D3 epimer and vitamin E in the diluted UCB plasma were below the limit of quantification, and could not be compared with UCB serum. Diluted UCB plasma can be used for the quantification of retinol and 25(OH)D3 by LC-MS/MS. By contrast, quantification of 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to limitations in analytical sensitivity. Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

  14. Quantification of coumarin in cinnamon and woodruff beverages using DIP-APCI-MS and LC-MS. (United States)

    Krieger, Sonja; Hayen, Heiko; Schmitz, Oliver J


    The use of the direct inlet probe-atmospheric-pressure chemical ionization (DIP-APCI) ion source developed in our laboratory coupled to a high resolution Q-TOF MS for the quantitative analysis of coumarin in different cinnamon samples was demonstrated in this study. Extraction of coumarin from various cinnamon samples was followed by DIP-APCI-mass spectrometry (MS) and liquid chromatography (LC)-MS analysis. For quantification, an external calibration with and without the use of stable isotope-labeled coumarin as internal standard was compared. The results obtained by DIP-APCI-MS and LC-MS were in good agreement. Even without the use of an internal standard satisfying linearity (R(2) > 0.997), recovery (94-104% for spiking levels between 100 and 5,000 mg/kg) and intra- and interday repeatability (2.2-13.8%RSD) was demonstrated using DIP-APCI-MS. To reduce the number of samples requiring quantitative analysis, the possibility of semi-quantitative screening of coumarin directly from powdered cinnamon using DIP-APCI-MS was shown. The analysis of woodruff-flavored beverages and cinnamon-flavored chewing gum by DIP-APCI-MS resulted in the formation of an artifact interfering with coumarin detection. As with other ambient ionization methods, special attention has to be paid to possible spectral interferences due to isobaric substances present in the sample matrix or formed from matrix components after ionization. The temperature-programmed vaporization in DIP-APCI-MS combined with the use of stable isotope-labeled coumarin as internal standard helped in recognizing this interference.

  15. LC-MS Based Serum Metabolomics for Identification of Hepatocellular Carcinoma Biomarkers in Egyptian Cohort (United States)

    Xiao, Jun Feng; Varghese, Rency S.; Zhou, Bin; Nezami Ranjbar, Mohammad R.; Zhao, Yi; Tsai, Tsung-Heng; Di Poto, Cristina; Wang, Jinlian; Goerlitz, David; Luo, Yue; Cheema, Amrita K.; Sarhan, Naglaa; Soliman, Hanan; Tadesse, Mahlet G.; Ziada, Dina Hazem; Ressom, Habtom W.


    confirmed significant differences between HCC and cirrhotic controls in metabolite levels of bile acid metabolites, long chain carnitines and small peptide. Our study provides useful insight into appropriate experimental design and computational methods for serum biomarker discovery using LC-MS/MS based metabolomics. This study has led to the identification of candidate biomarkers with significant changes in metabolite levels between HCC cases and cirrhotic controls. This is the first MS-based metabolic biomarker discovery study on Egyptian subjects that led to the identification of candidate metabolites that discriminate early stage HCC from patients with liver cirrhosis. PMID:23078175

  16. Organization of GC/MS and LC/MS metabolomics data into chemical libraries

    Directory of Open Access Journals (Sweden)

    DeHaven Corey D


    Full Text Available Abstract Background Metabolomics experiments involve generating and comparing small molecule (metabolite profiles from complex mixture samples to identify those metabolites that are modulated in altered states (e.g., disease, drug treatment, toxin exposure. One non-targeted metabolomics approach attempts to identify and interrogate all small molecules in a sample using GC or LC separation followed by MS or MSn detection. Analysis of the resulting large, multifaceted data sets to rapidly and accurately identify the metabolites is a challenging task that relies on the availability of chemical libraries of metabolite spectral signatures. A method for analyzing spectrometry data to identify and Quantify Individual Components in a Sample, (QUICS, enables generation of chemical library entries from known standards and, importantly, from unknown metabolites present in experimental samples but without a corresponding library entry. This method accounts for all ions in a sample spectrum, performs library matches, and allows review of the data to quality check library entries. The QUICS method identifies ions related to any given metabolite by correlating ion data across the complete set of experimental samples, thus revealing subtle spectral trends that may not be evident when viewing individual samples and are likely to be indicative of the presence of one or more otherwise obscured metabolites. Results LC-MS/MS or GC-MS data from 33 liver samples were analyzed simultaneously which exploited the inherent biological diversity of the samples and the largely non-covariant chemical nature of the metabolites when viewed over multiple samples. Ions were partitioned by both retention time (RT and covariance which grouped ions from a single common underlying metabolite. This approach benefitted from using mass, time and intensity data in aggregate over the entire sample set to reject outliers and noise thereby producing higher quality chemical identities. The

  17. Isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of ten steroid metabolites in serum

    DEFF Research Database (Denmark)

    Søeborg, Tue; Frederiksen, Hanne; Johannsen, Trine Holm


    An isotope-dilution TurboFlow-LC-MS/MS method for simultaneous quantification of the ten steroid metabolites dehydroepiandrosterone sulfate (DHEAS), progesterone, 17α-hydroxyprogesterone (17-OHP), Δ4-androstenedione (Adione), corticosterone, 11-deoxycortisol, cortisol, cortisone, testosterone (T...

  18. Understanding the regulation of estivation in a freshwater snail through iTRAQ-based comparative proteomics

    KAUST Repository

    Sun, Jin


    The apple snail Pomacea canaliculata is a freshwater gastropod with a remarkable ability to withstand seasonal or unpredictable dry conditions by entering estivation. Studies of P. canaliculata using conventional biochemical and the individual gene approaches have revealed the expressional changes of several enzymes and antioxidative genes in response to estivation and arousal. In this study, we applied iTRAQ-coupled two-dimensional LC-MS/MS to identify and quantify the global protein expression during the estivation and arousal of P. canaliculata. A total of 1040 proteins were identified, among which 701 proteins were quantified and compared across four treatments (i.e., control, active snails; short-term estivation, 3 days of exposure to air; prolonged estivation, 30 days of exposure to air; and arousal, 6 h after resubmergence in water) revealing 53 differentially expressed proteins. A comparison of protein expression profiles across treatments indicated that the proteome of this species was very insensitive to initial estivation, with only 9 proteins differentially expressed as compared with the control. Among the 9 proteins, the up-regulations of two immune related proteins indicated the initial immune response to the detection of stress cues. Prolonged estivation resulted in many more differentially expressed proteins (47 compared with short-term estivation treatment), among which 16 were down-regulated and 31 were up-regulated. These differentially expressed proteins have provided the first global picture of a shift in energy usage from glucose to lipid, prevention of protein degradation and elevation of oxidative defense, and production of purine for uric acid production to remove toxic ammonia during prolonged estivation in a freshwater snail. From prolonged estivation to arousal, only 6 proteins changed their expression level, indicating that access to water and food alone is not a necessary condition to reactivate whole-sale protein expression. A

  19. Improved LC-MS/MS of Heparan Sulfate Oligosaccharides via Chip-based Pulsed Make-up Flow


    Huang, Yu; Shi, Xiaofeng; Yu, Xiang; Leymarie, Nancy; Staples, Gregory O.; Yin, Hongfeng; Killeen, Kevin; Zaia, Joseph


    Microfluidic chip-based hydrophilic interaction chromatography (HILIC) is a useful separation system for liquid chromatography-mass spectrometry (LC-MS) in compositional profiling of heparan sulfate (HS) oligosaccharides; however, ions observed using HILIC LC-MS are low in charge. Tandem MS of HS oligosaccharide ions with low charge results in undesirable losses of SO3 from precursor ions during collision induced dissociation. One solution is to add metal cations to stabilize sulfate groups. ...

  20. Quantitative analysis of methyl and propyl parabens in neonatal DBS using LC-MS/MS. (United States)

    Yakkundi, Shirish; Mulla, Hussain; Pandya, Hitesh; Turner, Mark A; McElnay, James


    Excipients are used to overcome the chemical, physical and microbiological challenges posed by developing formulated medicines. Both methyl and propyl paraben are commonly used in pediatric liquid formulations. There is no data on systemic exposure to parabens in neonates. The European Study of Neonatal Exposure to Excipients project has investigated this. Results & methodology: DBS sampling was used to collect opportunistic blood samples. Parabens were extracted from the DBS and analyzed using a validated LC-MS/MS assay. The above assay was applied to analyze neonatal DBS samples. The blood concentrations of parabens in neonates confirm systemic exposure to parabens following administration of routine medicines.

  1. Larix decidua Bark as a Source of Phytoconstituents: An LC-MS Study

    Directory of Open Access Journals (Sweden)

    Valeria Baldan


    Full Text Available Larix decidua bark is a waste of the timber industry and is widely diffused in Northern Italy. This material can be considered a good source of antioxidants and phytoconstituents with possible use in cosmetic or nutraceutical products. In this study, simple extraction of larch bark was performed using mixtures of ethanol/water. Furthermore, the phytochemical composition of larch bark extract was studied using LC-MSn methods and the main constituents were identified as flavonoids, spiro-polyphenols, and procyanidins. To confirm the identification by LC-MS semi-preparative HPLC was performed in order to isolate the main constituents and verify the structures by 1H-NMR. Antioxidant properties were studied using an in vitro approach combining DPPH assay and LC-MS in order to establish different roles of the various classes of phytochemicasl of the extract. DPPH activity of some of the isolated compounds was also assessed. The overall results indicate this waste material as a good source of antioxidant compounds, mainly procyanidins, whichresulted the most active constituents in the DPPH assay.

  2. Expedient data mining for nontargeted high-resolution LC-MS profiles of biological samples. (United States)

    Hnatyshyn, Serhiy; Shipkova, Petia; Sanders, Mark


    The application of high-resolution LC-MS metabolomics for drug candidate toxicity screening reflects phenotypic changes of an organism caused by induced chemical interferences. Its success depends not only on the ability to translate the acquired analytical information into biological knowledge, but also on the timely delivery of the results to aid the decision making process in drug discovery and development. Recent improvements in analytical instrumentation have resulted in the ability to acquire extremely information-rich datasets. These new data collection abilities have shifted the bottleneck in the timeline of metabolomic studies to the data analysis step. This paper describes our approach to expedient data analysis of nontargeted high-resolution LC-MS profiles of biological samples. The workflow is illustrated with the example of metabolomics study of time-dependent fasting in male rats. The results from measurement of 220 endogenous metabolites in urine samples illustrate significant biochemical changes induced by fasting. The developed software enables the reporting of relative quantities of annotated components while maintaining practical turnaround times. Each component annotation in the report is validated using both calculated isotopic peaks patterns and experimentally determined retention time data on standards.

  3. [LC-MS/MS analysis of determination of strychnine and brucine in formaldehyde fixed tissue]. (United States)

    Zhan, Lan-fen; Liu, Ming-dong; Yan, You-yi; Ye, Yi; Wang, Wei; Wang, Zhi-hui; Zhao, Jun-hong; Liao, Lin-chuan


    To establish a method for determination of strychnine and brucine in formaldehyde fixed tissue by LC-MS/MS analysis. The samples were pretreated with solid phase extraction using SCX cartridges and separated on SB-C18 column with mobile phase 0.1% formic acid : 0.1% formic acid-acetonitrile (75:25). Electrospray ionization (ESI) source was utilized and operated in positive ion mode. Multiple reactions monitoring (MRM) mode was applied. External standard method was applied for quantitation. The chromatographic separation of strychnine and brucine in formaldehyde fixed nephritic and hepatic tissues resulted successfully. The standard curve was linear in the range of 0.002-2.0 microg/g for strychnine and brucine in formaldehyde fixed tissues, and the correlation coefficient was more than 0.996. The limits of detection (LOD) of strychnine and brucine in nephritic tissues were 0.06ng/g and 0.03 ng/g, respectively. The LOD of both chemicals were 0.3 ng/g in hepatic tissues. The extraction recovery rate was more than 74.5%. The precision of intra-day and inter-day were both less than 8.2%. Strychnine and brucine can be sensitive to be determined in formaldehyde fixed tissue by LC-MS/MS analysis. It can be applied in the forensic toxicological analysis.

  4. Mycotoxin production of Alternaria strains isolated from Korean barley grains determined by LC-MS/MS. (United States)

    Nguyen, Thuong T T; Kim, Jueun; Jeon, Sun Jeong; Lee, Chul Won; Magan, Naresh; Lee, Hyang Burm


    Twenty-four Alternaria strains were isolated from barley grain samples. These strains were screened for the production of mycotoxins on rice medium using thin layer chromatography. All 24 strains produced at least one of the five mycotoxins (ALT, AOH, ATX-I, AME, and TeA). Three representative strains, namely EML-BLDF1-4, EML-BLDF1-14, and EML-BLDF1-18, were further analyzed using a new LC-MS/MS-based mycotoxin quantification method. This method was used to detect and quantify Alternaria mycotoxins. We used positive ion electrospray mass spectrometry with multiple reaction mode (MRM) for the simultaneous quantification of various Alternaria mycotoxins produced by these strains. Five Alternaria toxins (ALT, ATX-I, AOH, AME, and TeA) were detected and quantified. Sample preparation included methanol extraction, concentration, and injection into LC-MS/MS. Limit of detection ranged from 0.13 to 4μg/mL and limit of quantification ranged from 0.25 to 8μg/mL. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Integrated GC-MS and LC-MS plasma metabonomics analysis of ankylosing spondylitis. (United States)

    Gao, Peng; Lu, Chen; Zhang, Fengxia; Sang, Ping; Yang, Dawei; Li, Xiang; Kong, Hongwei; Yin, Peiyuan; Tian, Jing; Lu, Xin; Lu, Aiping; Xu, Guowang


    Ankylosing spondylitis (AS) is a chronic inflammatory arthritis that predominantly affects the axial skeleton in adolescent patients. The natural history of the disease remains poorly characterized. In this study, we combined GC-MS and LC-MS techniques to evaluate the major metabolic changes in the plasma of AS patients in view of metabonomics. Univariate and multivariate analysis were employed for altered metabolite comparison and pattern recognition. Application of supervised partial least-squares discrminant analysis to either GC-MS or LC-MS data allowed accurate discrimination of AS patients from normal controls, demonstrating its potential diagnostic utilization. In addition, AS patients presented elevated plasma concentrations of proline, glucose, phosphate, urea, glycerol, phenylalanine and homocysteine but reduced levels of phosphocholines, tryptophan and a bipeptide - phenylalanyl-phenylalanine. In the context of their involved metabolic pathways, the identified metabolites were discussed accordingly. This investigation primarily proved that integrated chromatography-mass spectrometry and integrated uni- and multi-variate statistical analysis facilitated metabonomics to be a more promising tool in disease research.

  6. [Simultaneous determination of gestodene, etonogestrel and ethinylestradiol in plasma by LC-MS/MS following derivatization]. (United States)

    Liu, Xiao-Fen; Ding, Cun-Gang; Ge, Qing-Hua; Zhou, Zhen; Zhi, Xiao-Jin


    To establish a sensitive and specific method for simultaneous determination of gestodene, etonogestrel and ethinylestradiol in plasma by LC-MS/MS, plasma samples were extracted and derivatized before injection. An ESI ion source was used and operated in the positive ion mode with multiple reaction monitoring (MRM). Norgestrel was chosen as internal standard and performed on a C18 (100 mm x 2.1 mm, 5 microm) column. The concentrations of gestodene, etonogestrel and ethinylestradiol were measured, using step-gradient mobile phase and step-gradient flow rate. The method was validated over the concentration range of 0.1-20 ng x mL(-1) for gestodene and etonogestrel and 0.01-2 ng x mL(-1) for ethinylestradiol, and showed excellent linearity. The intra- and inter-assay accuracy and precision were below 10.0% and recovery was 93.6%-110.9% over the three concentration levels evaluated. The method was applied in pharmacokinetic study of the compound gestodene patch and the compound etonogestrel patch in rabbits. The LC-MS/MS method was selective, accurate and sensitive, especially the LOQ were 100 pg x mL(-1) for gestodene and etonogestrel and 10 pg x mL(-1) for ethinylestradiol. The method was successfully applied in pharmacokinetic study for contraceptives.

  7. [Effect investigation of coumarin constituents in Angelica dahurica on pharmacokinetics of docetaxel by LC-MS]. (United States)

    Guan, Yong-Mei; Zhu, Wei-Feng; Guan, Xue-Jing; Liao, Zheng-Gen; Zhao, Guo-Wei; Dong, Wei; Liang, Xin-Li


    The study was aimed to establish a liquid chromatography-tandem mass spectrometric method for the determination of the docetaxel concentration in rat plasma, and study the effect of coumarin constituents (imperatorin, isoimperatorin and oxypeucedanin) in Angelica dahurica on pharmacokinetics of docetaxel.Plasma was precipitated with acetonitrile and determined by LC-MS method with Paclitaxel as an internal standard. The specificity, linearity, range, accuracy, precision and stability of the method were suitable for the determination of docetaxel in plasma.Six sprague-dawley rats in each group received intragastric administration of docetaxel (50 mg•kg⁻¹), oxypeucedanin (8 mg•kg⁻¹) combined with docetaxel (50 mg•kg⁻¹), imperatorin (15 mg•kg⁻¹) combined with docetaxel (50 mg•kg⁻¹), and isoimperatorin(15 mg•kg⁻¹) combined with docetaxel (50 mg•kg⁻¹).Their drug plasma concentration was determined by LC-MS with Paclitaxel as an internal standard to draw plasma concentration-time curve, and the phamacokinetic parameters were calculated by DAS 2.0. The results showed that the phamacokinetic parameters of docetaxel all had notable changes when combined with imperatorin, isoimperatorin, and oxypeucedanin, respectively. The phamacokinetic parameters AUC and Cmax were significantly increased, indicating that coumarin constituents in Angelica dahurica could promote the oral bioavailability of docetaxel, and their effects were in the following order: oxypeucedanin> isoimperatorin> imperatorin. Copyright© by the Chinese Pharmaceutical Association.

  8. Analytical and clinical performance of the new Fujirebio 25-OH vitamin D assay, a comparison with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and three other automated assays. (United States)

    Saleh, Lanja; Mueller, Daniel; von Eckardstein, Arnold


    We evaluated the analytical and clinical performance of the new Lumipulse® G 25-OH vitamin D assay from Fujirebio, and compared it to a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and three other commercial automated assays. Total 25 hydroxy vitamin D (25(OH)D) levels were measured in 100 selected serum samples from our routine analysis with Fujirebio 25(OH)D assay. The results were compared with those obtained with LC-MS/MS and three other automated 25(OH)D assays (Abbott, Beckman, and Roche). The accuracy of each assay tested was evaluated against a Labquality reference serum panel for 25(OH)D (Ref!25OHD; University of Ghent). Intra- and inter-day imprecision of the Fujirebio 25(OH)D assay was Lumipulse G 25-OH vitamin D assay from Fujirebio demonstrated a good correlation with LC-MS/MS and some immunoassays. The performance of the assay is well-suited for routine 25(OH)D measurement in clinical serum samples. A correction for the observed negative bias vs. LC-MS/MS could be considered.

  9. Automated 2D peptide separation on a 1D nano-LC-MS system

    DEFF Research Database (Denmark)

    Taylor, Paul; Nielsen, Peter A; Trelle, Morten Beck


    Given the complexity of the mammalian proteome, high-resolution separation technologies are required to achieve comprehensive proteome coverage and to enhance the detection of low-abundance proteins. Among several technologies, Multidimensional Protein Identification Technology (MudPIT) enables...... the on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol, combining highly sensitive chromatography using a nanoflow liquid chromatography system (nano-LC) with a two......-dimensional precolumn in a vented column setup. When compared to the traditional MudPIT approach, this nanoflow variation demonstrated better first-phase separation leading to more proteins being characterized while using rather simple instrumentation and a protocol that requires less time and very little technical...

  10. Proteomics

    DEFF Research Database (Denmark)

    Dam, Svend; Stougaard, Jens


    Proteomics is an efficient tool to identify proteins present in specific tissues, cell types, or organelles. The resulting proteome reference maps and/or comparative analyses provide overviews of regulated proteins between wild type and mutants or between different conditions together...... proteomics data. Two characteristics of legumes are the high seed protein level and the nitrogen fixing symbiosis. Thus, the majority of the proteomics studies in Lotus have been performed on seed/pod and nodule/root tissues in order to create proteome reference maps and to enable comparative analyses within...... Lotus tissues or toward similar tissues from other legume species. More recently, N-glycan structures and compositions have been determined from mature Lotus seeds using glycomics and glycoproteomics, and finally, phosphoproteomics has been employed...

  11. Comparative proteomic analysis of hepatopancreas in Macrobrachium rosenbergii responded to Poly (I:C). (United States)

    Saray, Pheng; Roytrakul, Sittiruk; Pangeson, Tanapat; Phetrungnapha, Amnat


    Gel-enhanced liquid chromatography coupled with tandem mass spectrometry (GeLC-MS/MS) was used to analyze the proteome of Macrobrachium rosenbergii hepatopancreas responded to Poly (I:C). GeLC-MS/MS analysis identified 515 differentially-expressed proteins with ≥1.5 and ≤ -0.5 log 2 fold change. Of these, 195 differentially-expressed proteins were significantly matched to known proteins in the database, of which 102 proteins were up-regulated and 93 proteins were down-regulated. These proteins were classified into 21 categories, i.e. metabolic process, oxidative stress response, signaling, transcription, translation, cell cycle, transport, etc. Several immune factors were up-regulated upon Poly (I:C) injection. Protein-protein interaction network analysis of these immune factors identified three major protein clusters including RNAi, stress responses, and Toll pathway-proPO system, implying that Poly (I:C) activates immune responses in prawn through several mechanisms. Copyright © 2018. Published by Elsevier Ltd.

  12. Androgen and androgen metabolite levels in serum and urine of East African chimpanzees (Pan troglodytes schweinfurthii): comparison of EIA and LC-MS analyses. (United States)

    Preis, Anna; Mugisha, Lawrence; Hauser, Barbara; Weltring, Anja; Deschner, Tobias


    The primary male androgen testosterone (T) is often used as an endocrinological marker to investigate androgen-behaviour interactions in males. In chimpanzees and bonobos, studies investigating the relationship between T levels and dominance rank or aggressive behaviour have revealed contradictory results. The immunoassays used in these studies were originally developed for the measurement of steroids in serum. Their application to non-invasively collected samples, however, can lead to methodological problems due to cross-reacting metabolites, which might occur in urine or faeces but not in blood. The overall aim of this study, therefore, is to clarify whether a T enzyme immunoassay (EIA) is an applicable method to monitor testicular function in adult male chimpanzees. To estimate the impact of cross-reacting androgens on the used T EIA, we compared the results of an EIA measurement with a set of androgen metabolite levels measured by LC-MS. In urine from male chimpanzees, cross-reactivities appear to exist mainly with T and its exclusive metabolites, 5α-dihydrotestosterone (5α-DHT) and 5α-androstanediol (androstanediol). Both urinary and serum T levels of male chimpanzees were significantly higher than female T levels when measured with the T EIA, indicating a reliable measurement of testicular androgens and their exclusive metabolites with the used EIA. In urine from female chimpanzees, the comparison between LC-MS and T EIA results indicated a higher impact of cross-reactions with adrenal androgen metabolites. Therefore, the investigation of urinary T levels in female chimpanzees with a T EIA seems to be problematic. Overall our results show that a T EIA can be a reliable method to monitor testicular function in male chimpanzee urine and that LC-MS is a valuable tool for the validation of immunoassays. Copyright © 2011 Elsevier Inc. All rights reserved.

  13. The utility of qNMR to improve accuracy and precision of LC-MS bioanalysis

    Directory of Open Access Journals (Sweden)

    Dieter M. Drexler


    Full Text Available In LC-MS bioanalysis, samples and analytes are quantified against calibration solutions and curves which are derived via serial dilution from stock solutions that are prepared from dry reference standards. The analytical errors associated with the mass and volume measurements required for this preparation of stock solutions can culminate in a variance which might affect bioanalytical data accuracy. Especially in the case of extended studies with intermittent sample analysis, the multiple preparation of separate stock solutions can also adversely affect bioanalytical data precision. Discussed here is an illustrative case study where a single stock solution was utilized for a longitudinal study with multiple data points by means of an orthogonal and synergistic quality control via quantitative NMR methodology resulting in improved bioanalytical data.

  14. Analysis of Cell Metabolism Using LC-MS and Isotope Tracers. (United States)

    Mackay, Gillian M; Zheng, Liang; van den Broek, Niels J F; Gottlieb, Eyal


    Here we discuss our methods to analyze small polar compounds involved in central carbon metabolism using LC-MS. Methods described include sample extraction procedures for cells and medium, as well as for plasma/serum, urine, CSF, and tissue samples. Different extraction solvents are assessed. Our methods for using (13)C stable isotope tracers to examine the kinetics and distributions of mass isotopologues of many metabolites are discussed. Quantification methods are described for (13)C stable isotope tracer experiments as well as for unlabeled experiments. These methods were applied in a fumarate hydratase deficient cell model to show how isotope tracing can demonstrate shifts in metabolic pathways and, together with metabolite exchange rates, can be used to gain insights into changes in cell metabolism. © 2015 Elsevier Inc. All rights reserved.

  15. Current advances and strategies towards fully automated sample preparation for regulated LC-MS/MS bioanalysis. (United States)

    Zheng, Naiyu; Jiang, Hao; Zeng, Jianing


    Robotic liquid handlers (RLHs) have been widely used in automated sample preparation for liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis. Automated sample preparation for regulated bioanalysis offers significantly higher assay efficiency, better data quality and potential bioanalytical cost-savings. For RLHs that are used for regulated bioanalysis, there are additional requirements, including 21 CFR Part 11 compliance, software validation, system qualification, calibration verification and proper maintenance. This article reviews recent advances in automated sample preparation for regulated bioanalysis in the last 5 years. Specifically, it covers the following aspects: regulated bioanalysis requirements, recent advances in automation hardware and software development, sample extraction workflow simplification, strategies towards fully automated sample extraction, and best practices in automated sample preparation for regulated bioanalysis.

  16. Rugged Large Volume Injection for Sensitive Capillary LC-MS Environmental Monitoring

    Directory of Open Access Journals (Sweden)

    Hanne Roberg-Larsen


    Full Text Available A rugged and high throughput capillary column (cLC LC-MS switching platform using large volume injection and on-line automatic filtration and filter back-flush (AFFL solid phase extraction (SPE for analysis of environmental water samples with minimal sample preparation is presented. Although narrow columns and on-line sample preparation are used in the platform, high ruggedness is achieved e.g., injection of 100 non-filtrated water samples did not result in a pressure rise/clogging of the SPE/capillary columns (inner diameter 300 μm. In addition, satisfactory retention time stability and chromatographic resolution were also features of the system. The potential of the platform for environmental water samples was demonstrated with various pharmaceutical products, which had detection limits (LOD in the 0.05–12.5 ng/L range. Between-day and within-day repeatability of selected analytes were <20% RSD.

  17. [Determination of levodropropizine and its pharmacokinetics in human plasma using LC/MS/MS]. (United States)

    Zhao, Li-mei; Zhao, Li; Sun, Ya-xin; Qiu, Feng; Guo, Shan-bin


    To develop a rapid and sensitive LC/MS/MS method for the analysis of levodropropizine in plasma and study the pharmacokinetics of levodropropizine in healthy Chinese volunteers. Levodropropizine and zolmitriptan (internal standard, IS) were extracted from plasma samples and chromatographed on a C18 column and detected using a tandem mass spectrometer with a TurboIon Spray ionization interface. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions of the m/z 237 --> m/z 120 for levodropropizine and m/z 288 --> m/z 58 for the IS. The limit of quantification of the method for levodropropizine was 0.25 microg x L(-1). The assay was linear over the concentration range from 0.25 to 500.0 microg x L(-1) and intra- and inter-day precision over this range were levodropropizine.

  18. IDEOM: an Excel interface for analysis of LC-MS-based metabolomics data. (United States)

    Creek, Darren J; Jankevics, Andris; Burgess, Karl E V; Breitling, Rainer; Barrett, Michael P


    The application of emerging metabolomics technologies to the comprehensive investigation of cellular biochemistry has been limited by bottlenecks in data processing, particularly noise filtering and metabolite identification. IDEOM provides a user-friendly data processing application that automates filtering and identification of metabolite peaks, paying particular attention to common sources of noise and false identifications generated by liquid chromatography-mass spectrometry (LC-MS) platforms. Building on advanced processing tools such as mzMatch and XCMS, it allows users to run a comprehensive pipeline for data analysis and visualization from a graphical user interface within Microsoft Excel, a familiar program for most biological scientists. IDEOM is provided free of charge at, as a macro-enabled spreadsheet (.xlsb). Implementation requires Microsoft Excel (2007 or later). R is also required for full functionality. Supplementary data are available at Bioinformatics online.

  19. Quantitative measurement of DNA adducts using neutral hydrolysis and LC-MS. Validation of genotoxicity sensors. (United States)

    Tarun, Maricar; Rusling, James F


    Neutral hydrolysis and LC-MS/MS analysis of 6-nm-thick DNA-polyion films used in voltammetric genotoxicity screening sensors showed that concentrations of N7-guanine DNA adducts with methyl methanesulfonate and styrene oxide increased with incubation time with the same trends as found for sensor response. Results show that the genotoxicity sensors can be used to estimate relative DNA damage rates for chemical toxicity screening. Neutral thermal hydrolysis provided a relatively clean sample matrix allowing quantitative estimates of nucleobase adducts after several minutes of incubation with damage agents. In addition, an approximate standardization procedure for neutral thermal hydrolysis was developed and validated that avoids need for a pure standard and should be useful in cases where nucleobase adduct standards are unavailable or where their identities are unknown.

  20. Determination of mycotoxin exposure in Germany using an LC-MS/MS multibiomarker approach. (United States)

    Gerding, Johannes; Cramer, Benedikt; Humpf, Hans-Ulrich


    In this study, the exposure of a German population (n = 101) to mycotoxins was assessed using an LC-MS/MS urinary multibiomarker approach. Food consumption of the participants was documented with a food frequency questionnaire to correlate mycotoxin exposure with individual nutritional habits. The presence of 23 urinary biomarkers including trichothecenes (deoxynivalenol (DON), DON-3-glucuronide (DON-3-GlcA), T-2 toxin, HT-2 toxin (HT-2, HT-2-toxin-4-glucuronide (HT-2-GlcA), fumonisins (fumonisin B1, fumonisin B2), aflatoxins (aflatoxin B1, aflatoxin G2, aflatoxin B2, aflatoxin M1), zearalenone and derivatives (zearalanone, α-zearalenol, β-zearalenol, zearalenone-14-O-glucuronide, zearalanone-14-O-glucuronide, α-zearalenol-14-O-glucuronide/β-zearalenol-14-O-glucuronide), ochratoxin A, ochratoxin alpha, enniatin B and dihydrocitrinone was evaluated using a validated, sensitive "dilute and shoot"-LC-MS/MS method applying Scheduled MRM(TM) technology. Six mycotoxins and urinary metabolites were detected (DON, DON-3-GlcA, zearalenone-14-O-glucuronide, T-2 toxin, enniatin B, and dihydrocitrinone) in 87% of the samples in single- or co-occurence. Only DON and DON-3-GlcA were detectable in quantifiable amounts. A provisional mean daily intake of 0.52 μg DON/kg body weight was calculated. No statistical evidence for the correlation of staple food intake and urinary biomarker concentration could be determined. The results of this study suggest a low everyday exposure of the investigated German population to mycotoxins, but reveal peak exposures above the widely accepted tolerable daily intake to DON in parts of the population. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR

    International Nuclear Information System (INIS)

    Liu, Xinyue; St Ange, Kalib; Wang, Xiaohua; Lin, Lei; Zhang, Fuming


    relied on LC-MS and NMR analysis. • Monosaccharide compositional analysis relied on top-down NMR analysis. • Intact chain, oligosaccharide, and disaccharide analyses relied on LC-MS. • Differences due to parent heparin were observed using principal component analysis.

  2. Liquid-chromatography mass spectrometry (LC-MS) of steroid hormone metabolites and its applications (United States)

    Penning, Trevor M.; Lee, Seon-Hwa; Jin, Yi; Gutierrez, Alejandro; Blair, Ian A.


    Advances in liquid chromatography-mass spectrometry (LC-MS) can be used to measure steroid hormone metabolites in vitro and in vivo. We find that LC-Electrospray Ionization (ESI)-MS using a LCQ ion trap mass spectrometer in the negative ion mode can be used to monitor the product profile that results from 5α–dihydrotestosterone(DHT)-17β-glucuronide, DHT-17β-sulfate, and tibolone-17β-sulfate reduction catalyzed by human members of the aldo-keto reductase (AKR) 1C subfamily and assign kinetic constants to these reactions. We also developed a stable-isotope dilution LC-electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the quantitative analysis of estrone (E1) and its metabolites as pentafluorobenzyl (PFB) derivatives in human plasma in the attomole range. The limit of detection for E1-PFB was 740 attomole on column. Separations can be performed using normal-phase LC because ionization takes place in the gas phase rather than in solution. This permits efficient separation of the regioisomeric 2- and 4-methoxy-E1. The method was validated for the simultaneous analysis of plasma E2 and its metabolites: 2-methoxy-E2, 4-methoxy-E2, 16α-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16α-hydroxy-E1 from 5 pg/mL to 2,000 pg/mL. Our LC-MS methods have sufficient sensitivity to detect steroid hormone levels in prostate and breast tumors and should aid their molecular diagnosis and treatment. PMID:20083198

  3. LC-MS metabolomic analysis of environmental stressor impacts on the metabolite diversity in Nephthea spp.

    Directory of Open Access Journals (Sweden)

    Hedi Indra Januar


    Full Text Available Context: The soft coral Nephthea spp. is a source of terpenoid class that potentially has pharmaceutical properties. However, metabolite diversity and cytotoxic activity of this species are varied among coral reefs from various sites. Aim: To analyze the water quality in Nephthea spp. environment as a possible factor causing a difference in its metabolite diversity. Settings and Design: Nephthea spp. from seven sites were taken in October 2010 at the Alor District of Marine Protected Area, Indonesia. Materials and Methods: Water quality assessment was analyzed in situ and indexed by Canadian Council of Ministry Environment-Water Quality Index (CCME-WQI method. Meanwhile, metabolite diversity was analyzed by a LC-MS metabolomic method, using C18 reversed phase and gradient water-acetonitrile system. Statistical Analysis Used: Spearman′s rho and regression analysis were applied to correlate the water quality index to ecological index (richness, diversity, and evenness from LC-MS results. Results: The water quality index had a significant positive correlation and strong linear regression determinant to the total metabolite (R 2 = 0.704, particularly to semipolar metabolite richness (R 2 = 0.809, the area of terpenoid class in the organism. Conclusion: It can be concluded that water quality may serve as a major factor that affects the amount of richness in Nephthea spp. metabolites. When the water quality is lower, as environment stresses increases, it may affect the metabolite richness within direct disrupt of metabolite biosynthesis or indirect ecological means. Terpenoids are known as a soft coral antipredator (coral fishes, the amount of which depends on the water quality.

  4. 3D molecular cartography using LC-MS facilitated by Optimus and 'ili software. (United States)

    Protsyuk, Ivan; Melnik, Alexey V; Nothias, Louis-Felix; Rappez, Luca; Phapale, Prasad; Aksenov, Alexander A; Bouslimani, Amina; Ryazanov, Sergey; Dorrestein, Pieter C; Alexandrov, Theodore


    Our skin, our belongings, the world surrounding us, and the environment we live in are covered with molecular traces. Detecting and characterizing these molecular traces is necessary to understand the environmental impact on human health and disease, and to decipher complex molecular interactions between humans and other species, particularly microbiota. We recently introduced 3D molecular cartography for mapping small organic molecules (including metabolites, lipids, and environmental molecules) found on various surfaces, including the human body. Here, we provide a protocol and open-source software for 3D molecular cartography. The protocol includes step-by-step procedures for sample collection and processing, liquid chromatography-mass spectrometry (LC-MS)-based metabolomics, quality control (QC), molecular identification using MS/MS, data processing, and visualization with 3D models of the sampled environment. The LC-MS method was optimized for a broad range of small organic molecules. We enable scientists to reproduce our previously obtained results, and illustrate the broad utility of our approach with molecular maps of a rosemary plant and an ATM keypad after a PIN code was entered. To promote reproducibility, we introduce cartographical snapshots: files that describe a particular map and visualization settings, and that can be shared and loaded to reproduce the visualization. The protocol enables molecular cartography to be performed in any mass spectrometry laboratory and, in principle, for any spatially mapped data. We anticipate applications, in particular, in medicine, ecology, agriculture, biotechnology, and forensics. The protocol takes 78 h for a molecular map of 100 spots, excluding the reagent setup.

  5. Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xinyue, E-mail: [National Glycoengineering Research Center, Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology, State Key Laboratory of Microbial Technology, Shandong University, Jinan, Shandong, 250100 (China); Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); St Ange, Kalib, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Wang, Xiaohua, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); School of Computer and Information, Hefei University of Technology, Hefei (China); Lin, Lei, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); Zhang, Fuming, E-mail: [Department of Chemistry and Chemical Biology, Department of Chemical and Biological Engineering, Department of Biology, Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY, 12180 (United States); and others


    approach relied on LC-MS and NMR analysis. • Monosaccharide compositional analysis relied on top-down NMR analysis. • Intact chain, oligosaccharide, and disaccharide analyses relied on LC-MS. • Differences due to parent heparin were observed using principal component analysis.

  6. Characterisation of the metabolites of an antibacterial endophyte Botryodiplodia theobromae Pat. of Dracaena draco L. by LC-MS/MS. (United States)

    Zaher, Ahmed M; Moharram, Ahmad M; Davis, Richard; Panizzi, Peter; Makboul, Makboul A; Calderón, Angela I


    Botryodiplodia theobromae Pat. belongs to the endophytic fungi that live within the tissues of medicinal plants and produce bioactive natural products. The endophyte was isolated from the leaves of Dracaena draco L. The LC-MS-based metabolite fingerprinting of the ethyl acetate extract of B. theobromae with antibacterial activity led to the identification of 13 metabolites pertaining to various classes: dipeptides (maculosin and L,L-cyclo(leucylprolyl), alkaloid (norharman), coumarin and isocoumarins (bergapten, meranzin and monocerin), sesquiterpene (dihydrocumambrin A), aldehyde (formyl indanone), fatty alcohol (halaminol A) and fatty acid amide (palmitoleamide, palmitamide, capsi-amide and oleamide). This study reports for the first time, the LC-MS and LC-MS/MS identification of 13 known bioactive metabolites from the antibacterial ethyl acetate extract of B.theobromae isolated from the leaves of D. draco L.

  7. A sample preparation process for LC-MS/MS analysis of total protein drug concentrations in monkey plasma samples with antibody. (United States)

    Ji, Qin C; Rodila, Ramona; El-Shourbagy, Tawakol A


    The determination of protein concentrations in plasma samples often provides essential information in biomedical research, clinical diagnostics, and pharmaceutical discovery and development. Binding assays such as ELISA determine meaningful free analyte concentrations by using specific antigen or antibody reagents. Concurrently, mass spectrometric technology is becoming a promising complementary method to traditional binding assays. Mass spectrometric assays generally provide measurements of the total protein analyte concentration. However, it was found that antibodies may bind strongly with the protein analyte such that total concentrations cannot be determined. Thus, a sample preparation process was developed which included a novel "denaturing" step to dissociate binding between antibodies and the protein analyte prior to solid phase extraction of plasma samples and LC-MS/MS analysis. In so doing, the total protein analyte concentrations can be obtained. This sample preparation process was further studied by LC-MS analysis with a full mass range scan. It was found that the protein of interest and other plasma peptides were pre-concentrated, while plasma albumin was depleted in the extracts. This capability of the sample preparation process could provide additional advantages in proteomic research for biomarker discovery and validation. The performance of the assay with the novel denaturing step was further evaluated. The linear dynamic range was between 100.9ng/mL and 53920.0ng/mL with a coefficient of determination (r(2)) ranging from 0.9979 and 0.9997. For LLOQ and ULOQ samples, the inter-assay CV was 12.6% and 2.7% and inter-assay mean accuracies were 103.7% and 99.5% of theoretical concentrations, respectively. For QC samples, the inter-assay CV was between 2.1% and 4.9%, and inter-assay mean accuracies were between 104.1% and 110.0% of theoretical concentrations.

  8. Data-independent liquid chromatography/mass spectrometry (LC/MS(E)) detection and quantification of the secreted Apium graveolens pathogen defense protein mannitol dehydrogenase. (United States)

    Blackburn, Kevin; Cheng, Fang-Yi; Williamson, John D; Goshe, Michael B


    Plant cells secrete a wide variety of defense-related proteins into the extracellular space or apoplast in response to pathogen attack. One of these, mannitol dehydrogenase (MTD), is normally a cytoplasmic enzyme whose primary role is the regulation of intracellular levels of the sugar alcohol mannitol in plants. Recent immunological and biochemical evidence, however, suggests that MTD is also secreted into the apoplast in response to pathogen attack, despite lacking a known peptide signal sequence for Golgi-mediated secretion. Because many plant pathogenic fungi secrete mannitol to overcome pathogen-induced generation of reactive oxygen species (ROS) by the plant, extracellular localization of MTD is hypothesized to have a defensive role of catabolizing pathogen-secreted mannitol. In the current study, LC/MS(E) was used to analyze proteins in the secretome of Apium graveolens (celery) following treatment with salicylic acid (SA), an endogenous elicitor of defense responses in plants. Levels of MTD in the secretome of SA-treated celery cell cultures were found to be induced at least 18-fold over secretome samples from cell cultures not exposed to SA. This value is in close agreement with published immunological and biochemical observations. Overall, this report provides the first mass spectrometry identification and quantification measurements supporting the hypothesis that MTD is secreted in response to simulated pathogen attack via a non-classical secretion mechanism. As demonstrated with MTD secretion, LC/MS(E) can be implemented as a discovery-driven MRM-based quantitative approach which can be used to reveal potential post-translational modifications, thus providing a new method in the area of gel-free and label-free proteomic analysis. 2010 John Wiley & Sons, Ltd.

  9. Identification of plasma biomarkers for distinguishing bipolar depression from major depressive disorder by iTRAQ-coupled LC-MS/MS and bioinformatics analysis. (United States)

    Ren, Juanjuan; Zhao, Guoqing; Sun, Xiujia; Liu, Hongmei; Jiang, Ping; Chen, Jun; Wu, Zhiguo; Peng, Daihui; Fang, Yiru; Zhang, Chen


    It is important to differentiate between bipolar disorder (BD) and major depressive disorder (MDD) in the first depressive episode because of the potential treatment implications. Previous studies have mainly focused on the different clinical features or pathological biomarkers to distinguish these two diseases; however, a better understanding of the proteomics profiling of BD may help aid future therapeutic strategies. Here, we applied isobaric tags for relative and absolute quantification (iTRAQ) technology combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify differentially expressed proteins between MDD and bipolar depression (BP). In total, 30 MDD, 30 BP and 30 healthy subjects were included. Proteins from depleted plasma samples were digested into peptides, individually labeled with iTRAQ reagents, combined and subjected to LC-MS/MS and further bioinformatics analyses. Our results showed that 9 proteins were significantly altered between MDD and BP. Briefly, B2RAN2, B4E1B2, APOA1, ENG, SBSN and QSOX2 were up-regulated, whereas ORM1, MRC2 and SLPI were down-regulated. Most identified proteins were related to the immune system. The bioinformatics analysis showed that B2RAN2 (highly similar to vanin-1) was involved in the significantly enriched KEGG pathways "pantothenate and CoA biosynthesis" (P=0.009). B2RAN2 and ENG may play important roles in depression. They may serve as candidate biomarkers for distinguishing MDD and BP. Further validation and investigation are required to illuminate the roles of B2RAN2 and ENG in MDD and BP. The current study provided a potential and novel biomarker panel that may, in turn, aid the diagnosis of BD. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Determination of cystathionine beta-synthase activity in human plasma by LC-MS/MS: potential use in diagnosis of CBS deficiency.

    LENUS (Irish Health Repository)

    Krijt, Jakub


    Cystathionine β-synthase (CBS) deficiency is usually confirmed by assaying the enzyme activity in cultured skin fibroblasts. We investigated whether CBS is present in human plasma and whether determination of its activity in plasma could be used for diagnostic purposes. We developed an assay to measure CBS activity in 20 μL of plasma using a stable isotope substrate - 2,3,3-(2)H serine. The activity was determined by measurement of the product of enzyme reaction, 3,3-(2)H-cystathionine, using LC-MS\\/MS. The median enzyme activity in control plasma samples was 404 nmol\\/h\\/L (range 66-1,066; n = 57). In pyridoxine nonresponsive CBS deficient patients, the median plasma activity was 0 nmol\\/ho\\/L (range 0-9; n = 26), while in pyridoxine responsive patients the median activity was 16 nmol\\/hour\\/L (range 0-358; n = 28); this overlapped with the enzyme activity from control subject. The presence of CBS in human plasma was confirmed by an in silico search of the proteome database, and was further evidenced by the activation of CBS by S-adenosyl-L-methionine and pyridoxal 5\\'-phosphate, and by configuration of the detected reaction product, 3,3-(2)H-cystathionine, which was in agreement with the previously observed CBS reaction mechanism. We hypothesize that the CBS enzyme in plasma originates from liver cells, as the plasma CBS activities in patients with elevated liver aminotransferase activities were more than 30-fold increased. In this study, we have demonstrated that CBS is present in human plasma and that its catalytic activity is detectable by LC-MS\\/MS. CBS assay in human plasma brings new possibilities in the diagnosis of pyridoxine nonresponsive CBS deficiency.

  11. Improved stable isotope dilution assay for dietary folates using LC-MS/MS and its application to strawberries (United States)

    Striegel, Lisa; Chebib, Soraya; Netzel, Michael E.; Rychlik, Michael


    Folates play an important role in the human body and a deficiency of this vitamin can cause several diseases. Therefore, a reliable analytical method is crucial for the determination of folate vitamers in strawberries and other dietary folate sources. A stable isotope dilution LC-MS/MS method for analyzing folates in food was developed and validated. The folate vitamers Pteroylmonoglutamic acid, tetrahydrofolate, 5-methyltetrahydrofolate and 5-formyltetrahydrofolate were quantified using 13C-labelled internal standards. Validation of the assay was accomplished by determining linearity, precision, recovery, limit of detection and limit of quantification and revealed to be a precise, sensitive and accurate method to determine folate vitamers. Strawberries are worldwide consumed and known to be a good dietary source of nutritive compounds. Using this method, folate concentrations in selected commercial strawberry cultivars and experimental breeding lines grown in Germany were investigated. Total folates varied from 59 to 153 µg/100 g on fresh weight basis. Furthermore, folate content after lyophilizing or freezing did not show any significant differences compared to fresh strawberries. However, significant losses of total folates in pureed strawberries could be observed after 5 days of storage with only 16 % of the original concentration retained. In summary, some of the investigated strawberry cultivars/breeding lines can be considered as rich dietary sources of natural folates.

  12. Simultaneous determination of water-soluble vitamins in beverages and dietary supplements by LC-MS/MS. (United States)

    Kakitani, Ayano; Inoue, Tomonori; Matsumoto, Keiko; Watanabe, Jun; Nagatomi, Yasushi; Mochizuki, Naoki


    An LC-MS/MS method was developed for the simultaneous determination of 15 water-soluble vitamins that are widely used as additives in beverages and dietary supplements. This combined method involves the following simple pre-treatment procedures: dietary supplement samples were prepared by centrifugation and filtration after an extraction step, whereas beverage samples were diluted prior to injection. Chromatographic analysis in this method utilised a multi-mode ODS column, which provided reverse-phase, anion- and cation-exchange capacities, and therefore improved the retention of highly polar analytes such as water-soluble vitamins. Additionally, the multi-mode ODS column did not require adding ion pair reagents to the mobile phase. We optimised the chromatographic separation of 15 water-soluble vitamins by adjusting the mobile phase pH and the organic solvent. We also conducted an analysis of a NIST Standard Reference Material (SRM 3280 Multi-vitamin/Multi-element tablets) using this method to verify its accuracy. In addition, the method was applied to identify the vitamins in commercial beverages and dietary supplements. By comparing results with the label values and results obtained by official methods, it was concluded that the method could be used for quality control and to compose nutrition labels for vitamin-enriched products.

  13. Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis.

    Directory of Open Access Journals (Sweden)

    Angeles Cancino-Rodezno

    Full Text Available Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05 were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS, revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.

  14. Quantitative analysis of 11-nor-9-carboxy-tetrahydrocannbinol (THC-COOH) in urine by LC-MS/MS following a simple filtration. (United States)

    Rumpler, Marc J


    Quantification methods utilizing liquid chromatography tandem mass spectrometry (LC-MS/MS) are common in clinical and forensic toxicology laboratories and the efficiency and rapidity of such methods continues to evolve. In most cases, urine drug confirmation does not require a drug extraction and can quickly and easily be accomplished with a dilution followed by sample filtration. The report describes the validation of a simple confirmation method for 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH) and compared two types of filter extraction columns for sample clean-up. The method achieved a linear range of 10-3000ng/mL, acceptable bias (-4.7-2.6%) and precision (0.9-6.9%) and autosampler stability up to 72h. Universal filter columns offered less variable recovery over the linear range and fewer matrix interferences compared to THC-COOH specific filter columns. Authentic specimens testing positive for THC-COOH by LC-MS/MS were in good agreement with typically used GC-MS methods. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. LC-MS/MS versus TLC plus GC methods: Consistency of glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation. (United States)

    Jouhet, Juliette; Lupette, Josselin; Clerc, Olivier; Magneschi, Leonardo; Bedhomme, Mariette; Collin, Séverine; Roy, Sylvaine; Maréchal, Eric; Rébeillé, Fabrice


    Methods to analyze lipidomes have considerably evolved, more and more based on mass spectrometry technics (LC-MS/MS). However, accurate quantifications using these methods require 13C-labeled standards for each lipid, which is not feasible because of the very large number of molecules. Thus, quantifications rely on standard molecules representative of a whole class of lipids, which might lead to false estimations of some molecular species. Here, we determined and compared glycerolipid distributions from three different types of cells, two microalgae (Phaeodactylum tricornutum, Nannochloropsis gaditana) and one higher plant (Arabidopsis thaliana), using either LC-MS/MS or Thin Layer Chromatography coupled with Gas Chromatography (TLC-GC), this last approach relying on the precise quantification of the fatty acids present in each glycerolipid class. Our results showed that the glycerolipid distribution was significantly different depending on the method used. How can one reconcile these two analytical methods? Here we propose that the possible bias with MS data can be circumvented by systematically running in tandem with the sample to be analyzed a lipid extract from a qualified control (QC) of each type of cells, previously analyzed by TLC-GC, and used as an external standard to quantify the MS results. As a case study, we applied this method to compare the impact of a nitrogen deficiency on the three types of cells.

  16. SprayQc: a real-time LC-MS/MS quality monitoring system to maximize uptime using off the shelf components. (United States)

    Scheltema, Richard A; Mann, Matthias


    With the advent of high-throughput mass spectrometry (MS)-based proteomics, the magnitude and complexity of the performed experiments has increased dramatically. Likewise, investments in chromatographic and MS instrumentation are a large proportion of the budget of proteomics laboratories. Guarding measurement quality and maximizing uptime of the LC-MS/MS systems therefore requires constant care despite automated workflows. We describe a real-time surveillance system, called SprayQc, that continuously monitors the status of the peripheral equipment to ensure that operational parameters are within an acceptable range. SprayQc is composed of multiple plug-in software components that use computer vision to analyze electrospray conditions, monitor the chromatographic device for stable backpressure, interact with a column oven to control pressure by temperature, and ensure that the mass spectrometer is still acquiring data. Action is taken when a failure condition has been detected, such as stopping the column oven and the LC flow, as well as automatically notifying the appropriate operator. Additionally, all defined metrics can be recorded synchronized on retention time with the MS acquisition file, allowing for later inspection and providing valuable information for optimization. SprayQc has been extensively tested in our laboratory, supports third-party plug-in development, and is freely available for download from .

  17. MaXIC-Q Web: a fully automated web service using statistical and computational methods for protein quantitation based on stable isotope labeling and LC-MS. (United States)

    Tsou, Chih-Chiang; Tsui, Yin-Hao; Yian, Yi-Hwa; Chen, Yi-Ju; Yang, Han-Yin; Yu, Chuan-Yih; Lynn, Ke-Shiuan; Chen, Yu-Ju; Sung, Ting-Yi; Hsu, Wen-Lian


    Isotope labeling combined with liquid chromatography-mass spectrometry (LC-MS) provides a robust platform for analyzing differential protein expression in proteomics research. We present a web service, called MaXIC-Q Web (, for quantitation analysis of large-scale datasets generated from proteomics experiments using various stable isotope-labeling techniques, e.g. SILAC, ICAT and user-developed labeling methods. It accepts spectral files in the standard mzXML format and search results from SEQUEST, Mascot and ProteinProphet as input. Furthermore, MaXIC-Q Web uses statistical and computational methods to construct two kinds of elution profiles for each ion, namely, PIMS (projected ion mass spectrum) and XIC (extracted ion chromatogram) from MS data. Toward accurate quantitation, a stringent validation procedure is performed on PIMSs to filter out peptide ions interfered with co-eluting peptides or noise. The areas of XICs determine ion abundances, which are used to calculate peptide and protein ratios. Since MaXIC-Q Web adopts stringent validation on spectral data, it achieves high accuracy so that manual validation effort can be substantially reduced. Furthermore, it provides various visualization diagrams and comprehensive quantitation reports so that users can conveniently inspect quantitation results. In summary, MaXIC-Q Web is a user-friendly, interactive, robust, generic web service for quantitation based on ICAT and SILAC labeling techniques.

  18. A Collaborative Evaluation of LC-MS/MS Based Methods for BMAA Analysis: Soluble Bound BMAA Found to Be an Important Fraction

    Directory of Open Access Journals (Sweden)

    Elisabeth J. Faassen


    Full Text Available Exposure to β-N-methylamino-l-alanine (BMAA might be linked to the incidence of amyotrophic lateral sclerosis, Alzheimer’s disease and Parkinson’s disease. Analytical chemistry plays a crucial role in determining human BMAA exposure and the associated health risk, but the performance of various analytical methods currently employed is rarely compared. A CYANOCOST initiated workshop was organized aimed at training scientists in BMAA analysis, creating mutual understanding and paving the way towards interlaboratory comparison exercises. During this workshop, we tested different methods (extraction followed by derivatization and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS analysis, or directly followed by LC-MS/MS analysis for trueness and intermediate precision. We adapted three workup methods for the underivatized analysis of animal, brain and cyanobacterial samples. Based on recovery of the internal standard D3BMAA, the underivatized methods were accurate (mean recovery 80% and precise (mean relative standard deviation 10%, except for the cyanobacterium Leptolyngbya. However, total BMAA concentrations in the positive controls (cycad seeds showed higher variation (relative standard deviation 21%–32%, implying that D3BMAA was not a good indicator for the release of BMAA from bound forms. Significant losses occurred during workup for the derivatized method, resulting in low recovery (<10%. Most BMAA was found in a trichloroacetic acid soluble, bound form and we recommend including this fraction during analysis.

  19. Metabolomics Characterization of Two Apocynaceae Plants, Catharanthus roseus and Vinca minor, Using GC-MS and LC-MS Methods in Combination. (United States)

    Chen, Qi; Lu, Xueyan; Guo, Xiaorui; Guo, Qingxi; Li, Dewen


    Catharanthus roseus ( C. roseus ) and Vinca minor ( V. minor ) are two common important medical plants belonging to the family Apocynaceae. In this study, we used non-targeted GC-MS and targeted LC-MS metabolomics to dissect the metabolic profile of two plants with comparable phenotypic and metabolic differences. A total of 58 significantly different metabolites were present in different quantities according to PCA and PLS-DA score plots of the GC-MS analysis. The 58 identified compounds comprised 16 sugars, eight amino acids, nine alcohols and 18 organic acids. We subjected these metabolites into KEGG pathway enrichment analysis and highlighted 27 metabolic pathways, concentrated on the TCA cycle, glycometabolism, oligosaccharides, and polyol and lipid transporter (RFOS). Among the primary metabolites, trehalose, raffinose, digalacturonic acid and gallic acid were revealed to be the most significant marker compounds between the two plants, presumably contributing to species-specific phenotypic and metabolic discrepancy. The profiling of nine typical alkaloids in both plants using LC-MS method highlighted higher levels of crucial terpenoid indole alkaloid (TIA) intermediates of loganin, serpentine, and tabersonine in V. minor than in C. roseus . The possible underlying process of the metabolic flux from primary metabolism pathways to TIA synthesis was discussed and proposed. Generally speaking, this work provides a full-scale comparison of primary and secondary metabolites between two medical plants and a metabolic explanation of their TIA accumulation and phenotype differences.

  20. TLC-MS versus TLC-LC-MS fingerprints of herbal extracts. Part III. Application of the reversed-phase liquid chromatography systems with C18 stationary phase. (United States)

    Sajewicz, Mieczysław; Staszek, Dorota; Natic, Maja; Waksmundzka-Hajnos, Monika; Kowalska, Teresa


    In the previous paper from this series, we proposed mass spectrometric fingerprinting of complex botanical samples upon the examples of the pharmacologically important phenolic acids and flavonoids selectively extracted from Salvia lavandulifolia. In this study, we explore fingerprinting efficiency with a novel two-dimensional analytical system composed of the reversed-phase thin-layer chromatography and the reversed-phase high performance liquid chromatography with mass spectrometric detection (2D RP-TLC-RP-LC-MS). We also compare its efficiency with that of the one-dimensional analytical system (the reversed-phase thin-layer chromatography with mass spectrometric detection; 1D RP-TLC-MS). As our present study is basically focused on the method development, we considered it as justified to carry out our comparison with the phenolic acid extracts selectively derived from the Salvia lavandulifolia species, similar as it was done in Part II from this series. Upon the results obtained, it was established that the 1D RP-TLC-MS mode and the 2D RP-TLC-RP-LC-MS mode can be applied to fingerprinting of herbal extracts, and that the 2D RP-TLC-RP-LC mode can provide a more abundant information than that originating from the 1D RP-TLC mode.

  1. Selectivity in the sample preparation for the analysis of drug residues in products of animal origin using LC-MS

    NARCIS (Netherlands)

    Berendsen, B.J.A.; Stolker, A.A.M.; Nielen, M.W.F.


    Sample preparation is critical in relation to analysis time, sample throughput and therefore analysis costs. Due to recent advances in liquid chromatography-mass spectrometry (LC-MS) instrumentation, the detection of many compounds within one run became possible, and methods for the simultaneous

  2. LC-MS-SPE-NMR for the Isolation and Characterization of neo-Clerodane Diterpenoids from Teucrium luteum subsp. flavovirens

    NARCIS (Netherlands)

    Castro, A.; Moco, S.I.A.; Coll, J.; Vervoort, J.J.M.


    neo-Clerodane diterpenes of plant origin are molecules difficult to monitor due to their nonspecific UV/vis absorption. The present work describes for the first time the application of the LC-MS-SPE-NMR technique for the isolation and characterization of three new neo-clerodane diterpenes,

  3. Determination of Glyphosate Levels in Breast Milk Samples from Germany by LC-MS/MS and GC-MS/MS

    NARCIS (Netherlands)

    Steinborn, Angelika; Alder, Lutz; Michalski, Britta; Zomer, Paul; Bendig, Paul; Martinez, Sandra Aleson; Mol, Hans G.J.; Class, Thomas J.; Costa Pinheiro, Nathalie


    This study describes the validation and application of two independent analytical methods for the determination of glyphosate in breast milk. They are based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS), respectively. For

  4. Identification and Quantitative Analysis of Acetaminophen, Acetylsalicylic Acid, and Caffeine in Commercial Analgesic Tablets by LC-MS (United States)

    Fenk, Christopher J.; Hickman, Nicole M.; Fincke, Melissa A.; Motry, Douglas H.; Lavine, Barry


    An undergraduate LC-MS experiment is described for the identification and quantitative determination of acetaminophen, acetylsalicylic acid, and caffeine in commercial analgesic tablets. This inquiry-based experimental procedure requires minimal sample preparation and provides good analytical results. Students are provided sufficient background…

  5. Matrix effects break the LC behavior rule for analytes in LC-MS/MS analysis of biological samples (United States)

    High-performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) are generally accepted as the preferred techniques for detecting and quantitating analytes of interest in biological matrices on the basis of the rule that one chemical compound yields one LC-...

  6. Dried blood spot validation of five immunosuppressants, without hematocrit correction, on two LC-MS/MS systems

    NARCIS (Netherlands)

    Koster, Remco A; Veenhof, Herman; Botma, Rixt; Hoekstra, Alle Tjipke; Berger, Stefan P; Bakker, Stephan JL; Alffenaar, Jan-Willem C; Touw, Daan J


    AIM: Hematocrit (Ht) effects remain a challenge in dried blood spot (DBS) sampling. The aim was to develop an immunosuppressant DBS assay on two LC-MS/MS systems covering a clinically relevant Ht range without Ht correction. RESULTS: The method was partially validated for tacrolimus, sirolimus,

  7. Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM. (United States)

    Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav


    Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and

  8. Quantification of serotonin O-sulphate by LC-MS method in plasma of healthy volunteers.

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    Raimond eLozda


    Full Text Available The objective of this study was to test the hypothesis that serotonin O-sulphate (5 -HT-SO4 could be quantified in human plasma using modern liquid chromatography–mass spectrometry (LC-MS method as well as develop and validate that method. First, a suitable LC-MS method for detection of 5-HT-SO4 in human plasma samples was developed and validated. Second, a Pilot phase involving four healthy volunteers was executed, where a basal plasma level of 5-HT-SO4 was measured for all subjects and for one after the intake of 100 mg of a 5-hydroxytryptophan (5-HTP -containing food supplement used to promote serotonergic stimulation of the central nervous system. The basal level of 0.9 – 2.8 ng/mL of 5-HT-SO4 was observed. The changes of plasma 5HT-O-SO4 showed 1.2 ng/mL before and 22.6 ng/mL 1 h after stimulation. Finally, nine healthy volunteers were selected for the Study phase, where a basal plasma level of 5-HT-SO4 was measured before and after the intake of 5-HTP. One hour after stimulation, six study subjects showed a decrease in 5-HT-SO4 levels while three subjects showed an increase. The changes of plasma 5HT-O-SO4 from the Study phase showed an average 5-HT-SO4 level of 19.2 ng/mL before and 15.7 ng/mL 1 h after stimulation indicating ability of method to emphasise quantitative changes. This was the first study in which naturally occurring 5-HT-SO4 was detected in the samples of human plasma obtained from healthy volunteers. The method developed herein is specific to the measurement of 5-HT-SO4, sensitive enough to quantify intra-individual changes in the samples of plasma and opens up new possibilities to evaluate pathways of serotonin metabolism by minimally invasive methods. The discovery of novel biomarkers using such approaches is increasingly required to expedite development of mechanism-based therapeutics and patient stratification.

  9. Solvent optimization on Taxol extraction from Taxus baccata L., using HPLC and LC-MS

    Directory of Open Access Journals (Sweden)

    H Sadeghi-aliabadi


    Full Text Available "nBackground and the purpose of the study: Taxol, a natural antitumor agent, was first isolated from the extract of the bark of Taxus brevifolia Nutt., which is potentially a limited source for Taxol. In the search of an alternative source, optimum and cost benefit extracting solvents, various solvents with different percentage were utilized to extract Taxol from needles of Taxus baccata. "nMethods: One g of the dried needles of Taxus baccata, collected from Torkaman and Noor cities of Iran, was extracted with pure ethanol or acetone and 50% and 20% of ethanol or acetone in water. Solvents were evaporated to dryness and the residues were dissolved in 5 ml of methanol and filtered. To one ml of the filtrate was added 50 μl of cinamyl acetate as the internal standard and 20 μl of the resulting solution was subjected to the HPLC to determine the extraction efficiencies of tested solvents. Five μl of filtrate was also subjected to the LC-MS using water/acetonitrile (10/90 as mobile phase and applying positive electrospray ionization (ESI to identify the authenticity of Taxol. "nResults: Results of this study indicated that Taxol extraction efficiency was enhanced as the percentage of ethanol or acetone was increased. HPLC analysis showed that Taxol could be quantified by UV detection using standard curve. The standard curve covering the concentration ranges of 7.8 - 500 μg/ml was linear (r2= 0.9992 and CV% ranged from 0.52 to 15.36. LC-MS analysis using ESI in positive-ion mode confirmed the authenticity of Taxol (m/z 854; M+H, as well as some adduct ions such as M+Na (m/z 876, M+K (m/z 892 and M+CH3CN+H2O (m/z 913. "nConclusions: The results suggest that 100% acetone is the best solvent for the extraction of Taxol from Taxus baccata needles.

  10. Effect of trans fatty acid intake on LC-MS and NMR plasma profiles.

    Directory of Open Access Journals (Sweden)

    Gözde Gürdeniz

    Full Text Available The consumption of high levels of industrial trans fatty acids (TFA has been related to cardiovascular disease, diabetes and sudden cardiac death but the causal mechanisms are not well known. In this study, NMR and LC-MS untargeted metabolomics has been used as an approach to explore the impact of TFA intake on plasma metabolites.In a double-blinded randomized controlled parallel-group study, 52 overweight postmenopausal women received either partially hydrogenated soybean oil, providing 15.7 g/day of TFA (trans18:1 or control oil with mainly oleic acid for 16 weeks. Subsequent to the intervention period, the subjects participated in a 12-week dietary weight loss program. Before and after the TFA intervention and after the weight loss programme, volunteers participated in an oral glucose tolerance test. PLSDA revealed elevated lipid profiles with TFA intake. NMR indicated up-regulated LDL cholesterol levels and unsaturation. LC-MS profiles demonstrated elevated levels of specific polyunsaturated (PUFA long-chain phosphatidylcholines (PCs and a sphingomyelin (SM which were confirmed with a lipidomics based method. Plasma levels of these markers of TFA intake declined to their low baseline levels after the weight loss program for the TFA group and did not fluctuate for the control group. The marker levels were unaffected by OGTT.This study demonstrates that intake of TFA affects phospholipid metabolism. The preferential integration of trans18:1 into the sn-1 position of PCs, all containing PUFA in the sn-2 position, could be explained by a general up-regulation in the formation of long-chain PUFAs after TFA intake and/or by specific mobilisation of these fats into PCs. NMR supported these findings by revealing increased unsaturation of plasma lipids in the TFA group. These specific changes in membrane lipid species may be related to the mechanisms of TFA-induced disease but need further validation as risk markers.Registered at clinicaltrials

  11. Comparative proteomic analysis of early salt stress-responsive proteins in roots of SnRK2 transgenic rice

    Directory of Open Access Journals (Sweden)

    Nam Myung Hee


    Full Text Available Abstract Background The rice roots are highly salt-sensitive organ and primary root growth is rapidly suppressed by salt stress. Sucrose nonfermenting 1-related protein kinase2 (SnRK2 family is one of the key regulator of hyper-osmotic stress signalling in various plant cells. To understand early salt response of rice roots and identify SnRK2 signaling components, proteome changes of transgenic rice roots over-expressing OSRK1, a rice SnRK2 kinase were investigated. Results Proteomes were analyzed by two-dimensional electrophoresis and protein spots were identified by LC-MS/MS from wild type and OSRK1 transgenic rice roots exposed to 150 mM NaCl for either 3 h or 7 h. Fifty two early salt -responsive protein spots were identified from wild type rice roots. The major up-regulated proteins were enzymes related to energy regulation, amino acid metabolism, methylglyoxal detoxification, redox regulation and protein turnover. It is noted that enzymes known to be involved in GA-induced root growth such as fructose bisphosphate aldolase and methylmalonate semialdehyde dehydrogenase were clearly down-regulated. In contrast to wild type rice roots, only a few proteins were changed by salt stress in OSRK1 transgenic rice roots. A comparative quantitative analysis of the proteome level indicated that forty three early salt-responsive proteins were magnified in transgenic rice roots at unstressed condition. These proteins contain single or multiple potential SnRK2 recognition motives. In vitro kinase assay revealed that one of the identified proteome, calreticulin is a good substrate of OSRK1. Conclusions Our present data implicate that rice roots rapidly changed broad spectrum of energy metabolism upon challenging salt stress, and suppression of GA signaling by salt stress may be responsible for the rapid arrest of root growth and development. The broad spectrum of functional categories of proteins affected by over-expression of OSRK1 indicates that OSRK1

  12. GlycReSoft: a software package for automated recognition of glycans from LC/MS data.

    Directory of Open Access Journals (Sweden)

    Evan Maxwell

    Full Text Available Glycosylation modifies the physicochemical properties and protein binding functions of glycoconjugates. These modifications are biosynthesized in the endoplasmic reticulum and Golgi apparatus by a series of enzymatic transformations that are under complex control. As a result, mature glycans on a given site are heterogeneous mixtures of glycoforms. This gives rise to a spectrum of adhesive properties that strongly influences interactions with binding partners and resultant biological effects. In order to understand the roles glycosylation plays in normal and disease processes, efficient structural analysis tools are necessary. In the field of glycomics, liquid chromatography/mass spectrometry (LC/MS is used to profile the glycans present in a given sample. This technology enables comparison of glycan compositions and abundances among different biological samples, i.e. normal versus disease, normal versus mutant, etc. Manual analysis of the glycan profiling LC/MS data is extremely time-consuming and efficient software tools are needed to eliminate this bottleneck. In this work, we have developed a tool to computationally model LC/MS data to enable efficient profiling of glycans. Using LC/MS data deconvoluted by Decon2LS/DeconTools, we built a list of unique neutral masses corresponding to candidate glycan compositions summarized over their various charge states, adducts and range of elution times. Our work aims to provide confident identification of true compounds in complex data sets that are not amenable to manual interpretation. This capability is an essential part of glycomics work flows. We demonstrate this tool, GlycReSoft, using an LC/MS dataset on tissue derived heparan sulfate oligosaccharides. The software, code and a test data set are publically archived under an open source license.

  13. LC-MS/MS identification of the one-carbon cycle metabolites in human plasma. (United States)

    Gardner, Lidia A; Desiderio, Dominic M; Groover, Chassidy J; Hartzes, Anastasia; Yates, Charles R; Zucker-Levin, Audrey R; Bloom, Leonard; Levin, Michael C


    The one-carbon cycle is composed of four major biologically important molecules: methionine (L-Met), S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and homocysteine (Hcy). In addition to these key metabolites, there are multiple enzymes, vitamins, and cofactors that play essential roles in the cascade of the biochemical reactions that convert one metabolite into another in the cycle. Simultaneous quantitative measurement of four major metabolites can be used to detect possible aberrations in this vital cycle. Abnormalities in the one-carbon cycle might lead to hyper- or hypomethylation, homocystinemia, liver dysfunction, and accumulation of white-matter hyperintensities in the human brain. Previously published methods describe evaluation of several components of the one-carbon cycle, but none to our knowledge demonstrated simultaneous measurement of all four key molecules (L-Met, SAM, SAH, and Hcy). We describe a novel analytical method suitable for simultaneous identification and quantification of L-Met, SAM, SAH, and Hcy with LC-MS/MS. Moreover, we tested this method to identify these metabolites in human plasma collected from patients with multiple sclerosis and healthy individuals. In a pilot feasibility study, our results indicate that patients with multiple sclerosis showed abnormalities in the one-carbon cycle. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


    Directory of Open Access Journals (Sweden)

    Zinar Pinar GUMUS


    Full Text Available Cocaine is a powerfully addictive illicit drug. Cocaine abuse and addiction continue to increase in the world. Most analytical tests for the detection of cocaine use include the analysis of the metabolite, benzoylecgonine, in the urine. Benzoylecgonine is a major urinary metabolite of cocaine. Generally, the most common biologic matrices used for the analysis of cocaine contain the urine and blood however saliva was also added as a matrix in this study. Practical, quick, reliable, precise and accurate, reproducible analytical methods have been developed and validated for cocaine and benzoylecgonine. In addition, this chromatographic techniques were used as both initial test and confirmatory. The validated chromatographic method was successfully applied to the analysis of cocaine and benzoylecgonine compounds in synthetic biological matrix. Confirmation analyses were made by LC-MS/MS to support reliability of HPLC results. As a result of this study, it can be claimed that HPLC could be a good alternative for the analyses of various biological matrices in forensic studies. Also the matrices and concentrations were determined by HPLC instrument could be used where necessary.

  15. Determination of erythromycin and tylosin residues in honey by LC/MS/MS. (United States)

    Granja, Rodrigo; Niño, Alfredo Montes; Zucchetti, Roberto; Niño, Rosario Montes; Patel, Raj; Salerno, Alessandro Gonzalez


    Antibiotics are used in apiculture to protect bees against a variety of brood diseases. As a result of the development of resistance to oxytetracycline, erythromycin and tylosin are increasingly used for the prevention and treatment of these diseases. Therefore, Brazilian authorities have added these antibiotics to the National Regulatory Monitoring Program for the control of residues in honey. An analytical method has been developed for the determination of residues of erythromycin and tylosin in honey. The procedure involves solid-phase extraction of diluted honey samples with Bond Elut cartridges, followed by LC/MS with electrospray positive ionization in the multiple reaction monitoring mode. Two characteristic transitions were monitored for both drugs. Average analyte recoveries of erythromycin and tylosin ranged from 99 to 109% from sets of replicate honey samples fortified with drug concentrations of 5, 10, 15, and 20 microg/kg. The method decision limits were determined to be 1.27 and 0.59 microg/kg for erythromycin and tylosin, respectively. The detection capabilities were 5 and 5.2 microg/kg for erythromycin and tylosin, respectively.

  16. Multi-mycotoxin stable isotope dilution LC-MS/MS method for Fusarium toxins in beer. (United States)

    Habler, Katharina; Gotthardt, Marina; Schüler, Jan; Rychlik, Michael


    A stable isotope dilution LC-MS/MS multi-mycotoxin method was developed for 12 different Fusarium toxins including modified mycotoxins in beer (deoxynivalenol-3-glucoside, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyl-deoxynivalenol, HT2-toxin, T2-toxin, enniatin B, B1, A1, A, beauvericin and zearalenone). As sample preparation and purification of beer a combined solid phase extraction for trichothecenes, enniatins, beauvericin and zearalenone was firstly developed. The validation of the new method gave satisfying results: intra-day and inter-day precision and recoveries were 1-5%, 2-8% and 72-117%, respectively. In total, 61 different organic and conventional beer samples from Germany and all over the world were analyzed by using the newly developed multi-mycotoxin method. In summary, deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyldeoxynivaleneol and enniatin B were quantified in rather low contents in the investigated beer samples. None of the other monitored Fusarium toxins like 15-acetyldeoxynivalenol, HT2- and T2-toxin, zearalenone, enniatin B1, A1, A or beauvericin were detectable. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Library dependent LC-MS/MS acquisition via mzAPI/Live. (United States)

    Webber, James T; Askenazi, Manor; Ficarro, Scott B; Iglehart, Max A; Marto, Jarrod A


    The use of MS for characterization of small molecules, nucleotides, and proteins in model organisms as well as primary tissues and clinical samples continues to proliferate at a rapid pace. The complexity and dynamic range of target analytes in biological systems hinders comprehensive analysis and simultaneously drives improvements in instrument hardware and software. As a result, state-of-the-art commercial mass spectrometers are equipped with sophisticated embedded control systems that provide robust acquisition methods accessed through intuitive graphical interfaces. Although optimized for speed, these preconfigured scan functions are otherwise closed to end-user customization beyond simple, analytical-centric parameters supplied by the manufacturer. Here, we present an open-source framework (mzAPI/Live) that enables users to generate arbitrarily complex LC-MS(n) acquisition methods via simple Python scripting. As a powerful proof-of-concept, we demonstrate real-time assignment of tandem mass spectra through rapid query of NIST peptide libraries. This represents an unprecedented capability to make acquisition decisions based on knowledge of analyte structures determined during the run itself, thus providing a path toward biology-driven MS data acquisition for the broader community. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Determination of piroxicam from rat articular tissue and plasma based on LC-MS/MS. (United States)

    Kim, Han Sol; Cho, Ha Ra; Ho, Myoung Jin; Kang, Myung Joo; Choi, Yong Seok


    Osteoarthritis (OA) is the most common type of arthritis. To manage OA, in general, oral administration of non-steroidal anti-inflammatory drugs (NSAIDs) is used. Recently, the analgesic and anti-inflammatory efficacy of piroxicam (PX), a long-acting NSAID, by intra-articular (IA) administration in OA was reported, and the possibility that PX is distributed in articular tissues at a certain concentration was raised. Thus, herein, novel LC-MS/MS methods to detect PX in rat articular tissue and plasma are presented. For articular tissue, solvent extraction with acetonitrile for 12 h was employed and a protein precipitation method was used for the preparation of a plasma sample. The developed methods were validated by following the FDA guidelines, and the validated methods were successfully applied to a PK study of IA PX. The present study presents, to our knowledge, the first method of determining a drug in articular tissue. Additionally, the level of PX in articular tissue after IA PX administration was experimentally confirmed for the first time using the present methods. Therefore, the present methods provide a new direction for in vivo evaluation for IA PX formulations and contribute to the development of alternative IA PX formulations with better effects for the treatment of OA.

  19. A Greener, Quick and Comprehensive Extraction Approach for LC-MS of Multiple Mycotoxins

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    Andreas Breidbach


    Full Text Available In food/feed control, mycotoxin analysis is often still performed “one analyte at a time”. Here a method is presented which aims at making mycotoxin analysis environmentally friendlier through replacing acetonitrile by ethyl acetate and reducing chemical waste production by analyzing four mycotoxins together, forgoing sample extract clean-up, and minimizing solvent consumption. For this, 2 g of test material were suspended in 8 mL water and 16 mL ethyl acetate were added. Extraction was accelerated through sonication for 30 min and subsequent addition of 8 g sodium sulfate. After centrifugation, 500 µL supernatant were spiked with isotopologues, dried down, reconstituted in mobile phase, and measured with LC-MS. The method was validated in-house and through a collaborative study and the performance was fit-for-purpose. Repeatability relative standard deviation (RSDs between 16% at low and 4% at higher contaminations were obtained. The reproducibility RSDs were mostly between 12% and 32%. The trueness of results for T-2 toxin and Zearalenone were not different from 100%, for Deoxynivalenol and HT-2 toxin they were larger than 89%. The extraction was also adapted to a quick screening of Aflatoxin B1 in maize by flow-injection–mass spectrometry. Semi-quantitative results were obtained through standard addition and scan-based ion ratio calculations. The method proved to be a viable greener and quicker alternative to existing methods.

  20. Simultaneous determination of sweeteners in beverages by LC-MS/MS. (United States)

    Sakai, Hiroaki; Yamashita, Azusa; Tamura, Masayoshi; Uyama, Atsuo; Mochizuki, Naoki


    A new method was established for the simultaneous determination of 10 sweeteners and a degradation product in beverages by using LC-MS/MS. An ACQUITY UPLC BEH C18 (2.1 × 100 mm, 1.7 μm) was used as the LC column and 0.1% each of aqueous formic acid and formic acid in acetonitrile were used as the mobile phase. A simple and rapid determination of sweeteners was possible by diluting with a solvent, and in the case of some samples containing a large amount of foreign matter, after pre-treatment by diluting with solvent and clean-up of the sample using an Oasis HLB cartridge. All the validation results were satisfactory. As the regulations and standards for sweeteners vary from country to country, a field survey of 58 beverages marketed in Japan was performed using the present method. No issues concerning the labelling or food sanitation law were found in the tested samples.

  1. Quantification of pentose phosphate pathway (PPP) metabolites by liquid chromatography-mass spectrometry (LC-MS). (United States)

    Jannasch, Amber; Sedlak, Miroslav; Adamec, Jiri


    The pentose phosphate pathway plays an important role in several cellular processes including biosynthesis and catabolism of five-carbon sugars and generation of reducing power through NADPH synthesis. Although the pentose phosphate metabolic reaction network has been mapped in substantial detail, the comprehensive quantitative analysis of the rates and regulation of individual reactions remains a major interest for various biofields. Here we describe a simple method for comprehensive quantitative analysis of pentose phosphate pathway intermediates. The method is based on Group Specific Internal Standard Technology (GSIST) labeling in which an experimental sample and corresponding internal standards are derivatized in vitro with isotope-coded reagents in separate reactions, then mixed and analyzed in a single LC-MS run. The use of co-eluting isotope-coded internal standards and experimental molecules eliminates potential issues with ion suppression and allows for precise quantification of individual metabolites. Derivatization also increases hydrophobicity of the metabolites enabling their effective separation using reversed-phase chromatography.

  2. A High-Resolution LC-MS-Based Secondary Metabolite Fingerprint Database of Marine Bacteria

    KAUST Repository

    Lu, Liang


    © 2014 Macmillan Publishers Limited. All rights reserved. Marine bacteria are the most widely distributed organisms in the ocean environment and produce a wide variety of secondary metabolites. However, traditional screening for bioactive natural compounds is greatly hindered by the lack of a systematic way of cataloguing the chemical profiles of bacterial strains found in nature. Here we present a chemical fingerprint database of marine bacteria based on their secondary metabolite profiles, acquired by high-resolution LC-MS. Till now, 1,430 bacterial strains spanning 168 known species collected from different marine environments were cultured and profiled. Using this database, we demonstrated that secondary metabolite profile similarity is approximately, but not always, correlated with taxonomical similarity. We also validated the ability of this database to find species-specific metabolites, as well as to discover known bioactive compounds from previously unknown sources. An online interface to this database, as well as the accompanying software, is provided freely for the community to use.

  3. Multimycotoxin LC-MS/MS Analysis in Tea Beverages after Dispersive Liquid-Liquid Microextraction (DLLME). (United States)

    Pallarés, Noelia; Font, Guillermina; Mañes, Jordi; Ferrer, Emilia


    The aim of the present study was to develop a multimycotoxin liquid chromatography tandem mass spectrometry (LC-MS/MS) method with a dispersive liquid-liquid microextraction procedure (DLLME) for the analysis of AFs, 3aDON, 15aDON, NIV, HT-2, T-2, ZEA, OTA, ENNs, and BEA in tea beverages and to evaluate their mycotoxin contents. The proposed method was characterized in terms of linearity, limits of detection (LODs), limits of quantification (LOQs), recoveries, repeatability (intraday precision), reproducibility (interday precision), and matrix effects to check suitability. The results show LODs in the range of 0.05-10 μg/L, LOQs in the range of 0.2-33 μg/L, and recoveries in the range of 65-127% (RSD tea, red tea, green tea, and green mint tea. The results show that, of the analyzed mycotoxins, AFB2, AFG2, 15aDON, AFG1, and ENB were detected in the samples. AFB2 (14.4-32.2 μg/L) and 15aDON (60.5-61 μg/L) presented the highest levels. Green mint tea contained the highest concentration of mycotoxins. The risk assessment study shows that the population is not much exposed to mycotoxins through the consumption of tea beverages.

  4. New method for quantification of gasotransmitter hydrogen sulfide in biological matrices by LC-MS/MS (United States)

    Tan, Bo; Jin, Sheng; Sun, Jiping; Gu, Zhongkai; Sun, Xiaotian; Zhu, Yichun; Huo, Keke; Cao, Zonglian; Yang, Ping; Xin, Xiaoming; Liu, Xinhua; Pan, Lilong; Qiu, Furong; Jiang, Jian; Jia, Yiqun; Ye, Fuyuan; Xie, Ying; Zhu, Yi Zhun


    Hydrogen sulfide exists widely in mammalian tissues and plays a vital role in physiological and pathophysiological processes. However, striking differences with orders of magnitude were observed for the detected hydrogen sulfide concentrations in biological matrices among different measurements in literature, which lead to the uncertainty for examination the biological relevance of hydrogen sulfide. Here, we developed and validated a liquid chromatography- mass spectrometry (LC-MS/MS) method for the determination of hydrogen sulfide in various biological matrices by determination of a derivative of hydrogen sulfide and monobromobimane named sulfide dibimane (SDB). 36S-labeled SDB was synthesized and validated for using as an internal standard. This method has been successfully used to measure hydrogen sulfide levels in a broad range of biological matrices, such as blood, plasma, tissues, cells, and enzymes, across different species. Moreover, a novel mode that hydrogen sulfide could loosely and non-covalently bind to human serum protein (HSA) and hemoglobin (HB) was revealed by using the developed method. PMID:28406238

  5. [Analytical method of clofencet in animal fishery products by LC/MS]. (United States)

    Aoyagi, Mitsutoshi; Niiyama, Kazuhito; Nemoto, Satoru


    A method for the determination of clofencet in animal and fishery products was developed, using liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The sample was homogenized with water and hexane, and the homogenate was extracted with acetonitrile, then acetonitrile-water (4 : 1). An aliquot of the crude extract was passed through a C18 cartridge column (1,000 mg), and the eluate was concentrated. A solution of 10% sodium chloride and 1% sodium hydrogen carbonate, and ethyl acetate were added to the residue, and the mixture was shaken. After shaking, the aqueous phase was recovered and acidified with hydrochloric acid, and then clofencet was extracted with ethyl acetate. The extract was evaporated to dryness and the residue was dissolved in methanol-water (3 : 7). Clofencet was analyzed by LC/MS. The recoveries of clofencet from ten kinds of animal and fishery products were 77.8-97.8%, and the relative standard deviations were 0.6-5.8% (n=5). S/N of the peak of clofencet was >10 and no interfering peak was found in animal and fishery products fortified at 0.01 mg/kg.

  6. Analysis of cocaine/crack biomarkers in meconium by LC-MS. (United States)

    D'Avila, Felipe Bianchini; Ferreira, Pâmela C Lukasewicz; Salazar, Fernanda Rodrigues; Pereira, Andrea Garcia; Santos, Maíra Kerpel Dos; Pechansky, Flavio; Limberger, Renata Pereira; Fröehlich, Pedro Eduardo


    Fetal exposure to illicit drugs is a worldwide problem, since many addicted women do not stop using it during pregnancy. Cocaine consumed in powdered (snorted or injected) or smoked (crack cocaine) form are harmful for the baby and its side effects are not completely known. Meconium, the first stool of a newborn, is a precious matrix usually discarded, that may contain amounts of substances consumed in the last two trimesters of pregnancy. Analyzing this biological matrix it is possible to detect the unaltered molecule of cocaine (COC) or its metabolite benzoylecgonine (BZE) and pyrolytic products anhydroecgonine methyl ester (AEME) and anhydroecgonine (AEC). A liquid chromatography mass spectrometry (LC-MS) method was validated for meconium samples after solvent extraction, followed by direct injection of 10μL. Linearity covered a concentration range of 15 to 500ng/mg with a lower limit of quantification (LLOQ) of 15ng/mg for all analytes. Matrix effect was evaluated and showed adequate results. Detection of illicit substances usage can be crucial for the baby, since knowing that can help provide medical care as fast as possible. The method proved to be simple and fast, and was applied to 17 real meconium samples. Copyright © 2016 Elsevier B.V. All rights reserved.

  7. Comparison of Proteins in Whole Blood and Dried Blood Spot Samples by LC/MS/MS (United States)

    Chambers, Andrew G.; Percy, Andrew J.; Hardie, Darryl B.; Borchers, Christoph H.


    Dried blood spot (DBS) sampling methods are desirable for population-wide biomarker screening programs because of their ease of collection, transportation, and storage. Immunoassays are traditionally used to quantify endogenous proteins in these samples but require a separate assay for each protein. Recently, targeted mass spectrometry (MS) has been proposed for generating highly-multiplexed assays for biomarker proteins in DBS samples. In this work, we report the first comparison of proteins in whole blood and DBS samples using an untargeted MS approach. The average number of proteins identified in undepleted whole blood and DBS samples by liquid chromatography (LC)/MS/MS was 223 and 253, respectively. Protein identification repeatability was between 77 %-92 % within replicates and the majority of these repeated proteins (70 %) were observed in both sample formats. Proteins exclusively identified in the liquid or dried fluid spot format were unbiased based on their molecular weight, isoelectric point, aliphatic index, and grand average hydrophobicity. In addition, we extended this comparison to include proteins in matching plasma and serum samples with their dried fluid spot equivalents, dried plasma spot (DPS), and dried serum spot (DSS). This work begins to define the accessibility of endogenous proteins in dried fluid spot samples for analysis by MS and is useful in evaluating the scope of this new approach.

  8. Optimization and Evaluation Strategy of Esophageal Tissue Preparation Protocols for Metabolomics by LC-MS. (United States)

    Wang, Huiqing; Xu, Jing; Chen, Yanhua; Zhang, Ruiping; He, Jiuming; Wang, Zhonghua; Zang, Qingce; Wei, Jinfeng; Song, Xiaowei; Abliz, Zeper


    Sample preparation is a critical step in tissue metabolomics. Therefore, a comprehensive and systematic strategy for the screening of tissue preparation protocols is highly desirable. In this study, we developed an Optimization and Evaluation Strategy based on LC-MS to screen for a high-extractive efficiency and reproducible esophageal tissue preparation protocol for different types of endogenous metabolites (amino acids, carnitines, cholines, etc.), with a special focus on low-level metabolites. In this strategy, we first selected a large number of target metabolites based on literature survey, previous work in our lab, and known metabolic pathways. For these target metabolites, we tested different solvent extraction methods (biphasic solvent extraction, two-step [TS], stepwise [SW], all-in one [AO]; single-phase solvent extraction, SP) and esophageal tissue disruption methods (homogenized wet tissue [HW], ground wet tissue [GW], and ground dry tissue [GD]). A protocol involving stepwise addition of solvents and a homogenized wet tissue protocol (SWHW) was superior to the others. Finally, we evaluated the stability of endogenous metabolites in esophageal tissues and the sensitivity, reproducibility, and recovery of the optimal protocol. The results proved that the SWHW protocol was robust and adequate for bioanalysis. This strategy will provide important guidance for the standardized and scientific investigation of tissue metabolomics.

  9. Surveillance of drug abuse in Hong Kong by hair analysis using LC-MS/MS. (United States)

    Leung, K Wing; Wong, Zack C F; Ho, Janet Y M; Yip, Ada W S; Cheung, Jerry K H; Ho, Karen K L; Duan, Ran; Tsim, Karl W K


    The aim of this study is to reveal the habits of drug abusers in hair samples from drug rehabilitation units in Hong Kong. With the application of liquid chromatography-tandem mass spectrometry (LC-MS/MS) technology, a total of 1771 hair samples were analyzed during the period of hair testing service (January 2012 to March 2016) provided to 14 drug rehabilitation units including non-governmental organizations (NGOs), rehabilitation centers, and medical clinics. Hair samples were analyzed for abused drugs and their metabolites simultaneously, including ketamine, norketamine, cocaine, benzoylecgonine, cocaethylene, norcocaine, codeine, MDMA, MDA, MDEA, amphetamine, methamphetamine, morphine, 6-acetylmorphine, phencyclidine, and methadone. The results showed that ketamine (77.2%), cocaine (21.3%), and methamphetamine (16.5%) were the frequently detected drugs among those drug abusers, which is consistent with the reported data. In addition, the usage of multiple drugs was also observed in the hair samples. About 29% of drug-positive samples were detected with multiple drug use. Our studies prove that our locally developed hair drug-testing method and service can be a valid tool to monitor the use of abused drugs, and which could facilitate rehabilitation program management. Copyright © 2017 John Wiley & Sons, Ltd.

  10. Determination of raloxifene hydrochloride in human urine by LC-MS-MS

    Directory of Open Access Journals (Sweden)

    K. Tharpa


    Full Text Available A sensitive and selective liquid chromatographic-tandem mass spectrometric (LC-MS-MS method was developed to determine raloxifene hydrochloride (RLX in human urine. After a solid-phase extraction with SPE cartridge, the urine sample was analyzed on a C18 column (Symmetry 3.5μm; 50 mm4.6 mm i.d interfaced with a triple quadruple tandem mass spectrometer. A positive electrospray ionization was employed as the ionization source. The mobile phase consisted of ammonium acetate (pH 4.0–acetonitrile (60:40, v/v.The method was linear over a concentration range of 20–1000 ng mL-1. The lower limit of quantitation was 20 ng mL-1. The intra-day and inter-day relative standard deviation across three validation runs over the entire concentration range was <10.5%. The accuracy determined at three concentrations (50, 500 and 850 ng mL-1 RLX was within ±0.84% in terms of relative errors.

  11. LC-MS-MS quantitative analysis reveals the association between FTO and DNA methylation.

    Directory of Open Access Journals (Sweden)

    Yuting Zhu

    Full Text Available Fat mass and obesity-associated protein (FTO is α-ketoglutarate-dependent dioxygenase and responsible for demethylating N6-methyladenosine (m6A in mRNA, 3-methylthymine (m3T in single-stranded DNA (ssDNA and 3-methyluracil (m3U in single-stranded RNA (ssRNA. Its other function remains unknown but thousands of mammalian DNA show 5-methyl-2'-deoxycytidine (5mdC modification and 5mdC demethylases are required for mammalian energy homeostasis and fertility. Here, we aimed to confirm whether FTO proteins can demethylate 5mdC in DNA. However, we found that FTO exhibits no potent demethylation activity against 5mdC in vitro and in vivo by using liquid chromatography-tandem mass spectrometry (LC-MS-MS. The result showed FTO demethylase has the characteristics of high substrates specificity and selectivity. In addition, we also used immunofluorescence technique to demonstrate overexpression of wild type TET2, but not FTO and mutant TET2 in Hela cells results in higher levels of 5-hydroxymethyl-2'-deoxycytidine (5hmdC generated from 5mdC. In conclusion, our results not only reveal the enzymatic activity of FTO, but also may facilitate the future discovery of proteins involved in epigenetic modification function.

  12. Analyses of marketplace tacrolimus drug product quality: bioactivity, NMR and LC-MS. (United States)

    Sommers, Cynthia D; Pang, Eric S; Ghasriani, Houman; Berendt, Robert T; Vilker, Vincent L; Keire, David A; Boyne, Michael T


    Tacrolimus (FK506) is a potent, narrow therapeutic index, immunosuppressive drug used to avoid organ rejection in patients that have undergone organ transplantation. Recent clinical reports suggested a significant reduction in the tacrolimus concentration/dose ratio in the plasma of liver and kidney recipients when the reference listed drug was substituted with a generic drug. In response to these concerns about switching between tacrolimus from different approved manufacturers during treatment, the FDA initiated purity, potency and quality studies of the innovator and generic tacrolimus products available in the US marketplace. A combination of analytical methods, including mass spectrometry (LC-MS), nuclear magnetic resonance (NMR) and bioactivity assay were developed and validated to assess the quality of tacrolimus. These tests measured the identity, impurities and activity of tacrolimus from active pharmaceutical ingredient (API) sources and with formulated drug product from five different approved manufactures. In addition, some testing was performed on tacrolimus capsules obtained from a non US approved Indian source. The data obtained showed no discernible difference in the impurity profiles and potency between the generic and innovator tacrolimus products. Copyright © 2013. Published by Elsevier B.V.

  13. Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS. (United States)

    Sinclair, John; Timms, John F


    Blood-borne biomarkers are urgently required for the early detection, accurate diagnosis and prognosis of disease. Additionally, improved methods of profiling serum and plasma proteins for biomarker discovery efforts are needed. Herein, we report a quantitative method based on amino-group labelling of serum proteins (rather than peptides) with isobaric tandem mass tags (TMT) and incorporating immune-based depletion, gel-based and strong anion exchange separation of proteins prior to differential endoproteinase treatment and liquid chromatography tandem mass spectrometry. We report a generally higher level of quantitative coverage of the serum proteome compared to other peptide-based isobaric tagging approaches and show the potential of the method by applying it to a set of unique samples that pre-date the diagnosis of pancreatic cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Glycoproteomics: identifying the glycosylation of prostate specific antigen at normal and high isoelectric points by LC-MS/MS. (United States)

    Song, Ehwang; Mayampurath, Anoop; Yu, Chuan-Yih; Tang, Haixu; Mechref, Yehia


    Prostate specific antigen (PSA) is currently used as a biomarker to diagnose prostate cancer. PSA testing has been widely used to detect and screen prostate cancer. However, in the diagnostic gray zone, the PSA test does not clearly distinguish between benign prostate hypertrophy and prostate cancer due to their overlap. To develop more specific and sensitive candidate biomarkers for prostate cancer, an in-depth understanding of the biochemical characteristics of PSA (such as glycosylation) is needed. PSA has a single glycosylation site at Asn69, with glycans constituting approximately 8% of the protein by weight. Here, we report the comprehensive identification and quantitation of N-glycans from two PSA isoforms using LC-MS/MS. There were 56 N-glycans associated with PSA, whereas 57 N-glycans were observed in the case of the PSA-high isoelectric point (pI) isoform (PSAH). Three sulfated/phosphorylated glycopeptides were detected, the identification of which was supported by tandem MS data. One of these sulfated/phosphorylated N-glycans, HexNAc5Hex4dHex1s/p1 was identified in both PSA and PSAH at relative intensities of 0.52 and 0.28%, respectively. Quantitatively, the variations were monitored between these two isoforms. Because we were one of the laboratories participating in the 2012 ABRF Glycoprotein Research Group (gPRG) study, those results were compared to that presented in this study. Our qualitative and quantitative results summarized here were comparable to those that were summarized in the interlaboratory study.


    Method 544 is an accurate and precise analytical method to determine six microcystins (including MC-LR) and nodularin in drinking water using solid phase extraction and liquid chromatography tandem mass spectrometry (SPE-LC/MS/MS). The advantage of this SPE-LC/MS/MS is its sensi...

  16. Autophagy is highly targeted among host comparative proteomes during infection with different virulent RABV strains. (United States)

    Li, Ling; Jin, Hongli; Wang, Hualei; Cao, Zengguo; Feng, Na; Wang, Jianzhong; Zhao, Yongkun; Zheng, Xuexing; Hou, Pengfei; Li, Nan; Chi, Hang; Huang, Pei; Jiao, Cuicui; Li, Qian; Wang, Lina; Wang, Tiecheng; Sun, Weiyang; Gao, Yuwei; Tu, Changchun; Hu, Guixue; Yang, Songtao; Xia, Xianzhu


    Rabies virus (RABV) is a neurotropic virus that causes serious disease in humans and animals worldwide. It has been reported that different RABV strains can result in divergent prognoses in animal model. To identify host factors that affect different infection processes, a kinetic analysis of host proteome alterations in mouse brains infected with different virulent RABV strains was performed using isobaric tags for a relative and absolute quantification (iTRAQ)-liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics approach, and this analysis identified 147 differentially expressed proteins (DEPs) between the pathogenic challenge virus standard (CVS)-11 strain and the attenuated SRV9 strain. Bioinformatics analyses of these DEPs revealed that autophagy and several pathways associated with autophagy, such as mammalian target of rapamycin (mTOR) signaling, p70S6K signaling, nuclear factor erythroid 2-related factor 2 (NRF2)-mediated oxidative stress and superoxide radical degradation, were dysregulated. Validation of the proteomic data showed that attenuated SRV9 induced more autophagosome accumulation than CVS-11 in an in vitro model. Our findings provide new insights into the pathogenesis of RABV and encourage further studies on this topic.

  17. Profiles of Steroid Hormones in Canine X-Linked Muscular Dystrophy via Stable Isotope Dilution LC-MS/MS. (United States)

    Martins-Júnior, Helio A; Simas, Rosineide C; Brolio, Marina P; Ferreira, Christina R; Perecin, Felipe; Nogueira, Guilherme de P; Miglino, Maria A; Martins, Daniele S; Eberlin, Marcos N; Ambrósio, Carlos E


    Golden retriever muscular dystrophy (GRMD) provides the best animal model for characterizing the disease progress of the human disorder, Duchenne muscular dystrophy (DMD). The purpose of this study was to determine steroid hormone concentration profiles in healthy golden retriever dogs (control group - CtGR) versus GRMD-gene carrier (CaGR) and affected female dogs (AfCR). Therefore, a sensitive and specific analytical method was developed and validated to determine the estradiol, progesterone, cortisol, and testosterone levels in the canine serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). To more accurately understand the dynamic nature of the serum steroid profile, the fluctuating levels of these four steroid hormones over the estrous cycle were compared across the three experimental groups using a multivariate statistical analysis. The concentration profiles of estradiol, cortisol, progesterone, and testosterone revealed a characteristic pattern for each studied group at each specific estrous phase. Additionally, several important changes in the serum concentrations of cortisol and estradiol in the CaGR and AfCR groups seem to be correlated with the status and progression of the muscular dystrophy. A comprehensive and quantitative monitoring of steroid profiles throughout the estrous cycle of normal and GRMD dogs were achieved. Significant differences in these profiles were observed between GRMD and healthy animals, most notably for estradiol. These findings contribute to a better understanding of both dog reproduction and the muscular dystrophy pathology. Our data open new venues for hormonal behavior studies in dystrophinopathies and that may affect the quality of life of DMD patients.

  18. Profiles of Steroid Hormones in Canine X-Linked Muscular Dystrophy via Stable Isotope Dilution LC-MS/MS.

    Directory of Open Access Journals (Sweden)

    Helio A Martins-Júnior

    Full Text Available Golden retriever muscular dystrophy (GRMD provides the best animal model for characterizing the disease progress of the human disorder, Duchenne muscular dystrophy (DMD. The purpose of this study was to determine steroid hormone concentration profiles in healthy golden retriever dogs (control group - CtGR versus GRMD-gene carrier (CaGR and affected female dogs (AfCR. Therefore, a sensitive and specific analytical method was developed and validated to determine the estradiol, progesterone, cortisol, and testosterone levels in the canine serum by isotope dilution liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS. To more accurately understand the dynamic nature of the serum steroid profile, the fluctuating levels of these four steroid hormones over the estrous cycle were compared across the three experimental groups using a multivariate statistical analysis. The concentration profiles of estradiol, cortisol, progesterone, and testosterone revealed a characteristic pattern for each studied group at each specific estrous phase. Additionally, several important changes in the serum concentrations of cortisol and estradiol in the CaGR and AfCR groups seem to be correlated with the status and progression of the muscular dystrophy. A comprehensive and quantitative monitoring of steroid profiles throughout the estrous cycle of normal and GRMD dogs were achieved. Significant differences in these profiles were observed between GRMD and healthy animals, most notably for estradiol. These findings contribute to a better understanding of both dog reproduction and the muscular dystrophy pathology. Our data open new venues for hormonal behavior studies in dystrophinopathies and that may affect the quality of life of DMD patients.

  19. Comparison of clonazepam compliance by measurement of urinary concentration by immunoassay and LC-MS/MS in pain management population. (United States)

    West, Robert; Pesce, Amadeo; West, Cameron; Crews, Bridgit; Mikel, Charles; Almazan, Perla; Rosenthal, Murray; Latyshev, Sergey


    Physicians determine patient compliance with their medications by use of urine drug testing. It is known that measurement of benzodiazepines is limited by immunoassay specificity and cutoff limits and therefore does not offer physicians an accurate picture of their patients' compliance with these medications. A few studies have used lower cutoffs to demonstrate patient compliance. To define more appropriate cutoffs for compliance monitoring of patients prescribed clonazepam as determined using immunoassay and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A diagnostic accuracy study of the urinary excretion of clonazepam. Millennium Laboratories performed measurements on the urinary excretion of pain patients prescribed clonazepam as the indicator test. This benzodiazepine was chosen because it forms one major metabolite, 7-aminoclonazepam which is specific for that drug. Patients whose only benzodiazepine medication was clonazepam were selected as the test population. The Millennium Laboratories test database was filtered first to select patients on clonazepam, then a second filter was used to eliminate patients with any other listed benzodiazepine medications. Samples were tested using the Microgenics DRI benzodiazepine assay with a 200 ng/mL cutoff. The same samples were quantitatively assessed for 7-aminoclonazepam by LC-MS/MS with a cutoff of 40 ng/mL. The results from the immunoassay were scored as positive or negative while the quantitative results from the LC-MS/MS were also scored as positive or negative depending upon their concentration. Samples from 180 patients met these medication criteria. The positivity rates were 21% (38 samples) by immunoassay. The positivity rate was 70% (126 samples) if the LC-MS/MS cutoff was set at 200 ng/mL. However, the positivity rate was 87% (157 samples) if the LC-MS/MS was set at 40 ng/mL. Concentration distributions revealed a significant fraction (7%) in the 40 - 100 ng/mL range. A limitation of the study

  20. Proteomics dataset

    DEFF Research Database (Denmark)

    Bennike, Tue Bjerg; Carlsen, Thomas Gelsing; Ellingsen, Torkell


    The datasets presented in this article are related to the research articles entitled “Neutrophil Extracellular Traps in Ulcerative Colitis: A Proteome Analysis of Intestinal Biopsies” (Bennike et al., 2015 [1]), and “Proteome Analysis of Rheumatoid Arthritis Gut Mucosa” (Bennike et al., 2017 [2...... conducted the sample preparation and liquid chromatography mass spectrometry (LC-MS/MS) analysis of all samples in one batch, enabling label-free comparison between all biopsies. The datasets are made publicly available to enable critical or extended analyses. The proteomics data and search results, have...... been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifiers PXD001608 for ulcerative colitis and control samples, and PXD003082 for rheumatoid arthritis samples....

  1. Deep coverage of the beer proteome. (United States)

    Grochalová, Martina; Konečná, Hana; Stejskal, Karel; Potěšil, David; Fridrichová, Danuše; Srbová, Eva; Ornerová, Kateřina; Zdráhal, Zbyněk


    We adopted an approach based on peptide immobilized pH gradient-isoelectric focusing (IPG-IEF) separation, coupled with LC-MS/MS, in order to maximize coverage of the beer proteome. A lager beer brewed using traditional Czech technology was degassed, desalted and digested. Tryptic peptides were separated by isoelectric focusing on an immobilized pH gradient strip and, after separation, the gel strip was divided into seven equally sized parts. Peptides extracted from gel fractions were analyzed by LC-MS/MS. This approach resulted in a three-fold increase in the number of proteins identified (over 1700) when compared to analysis of unfractionated beer processed by a filter-aided sample preparation (FASP). Over 1900 protein groups (PGs) in total were identified by both approaches. The study significantly extends knowledge about the beer proteome and demonstrates its complexity. Detailed knowledge of the protein content, especially gluten proteins, will enhance the evaluation of potential health risks related to beer consumption (coeliac disease) and will contribute to improving beer quality. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC-MS. (United States)

    Di, Li; Kerns, Edward H; Li, Susan Q; Carter, Guy T


    The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction potential was investigated, in order to have evidence for selecting a reliable in vitro CYP450 inhibition assay to support drug discovery. Three assays were studied: individual rhCYP isozymes and corresponding coumarin derivative-probe substrates with fluorescent detection, human liver microsomes (HLM) and cocktail drug-probe substrates with LC-MS detection, and double cocktail rhCYP isozymes mix and drug-probe mix with LC-MS detection. Data comparisons showed that the rhCYP-fluorescent assay and the cocktail assay with HLM-LC-MS had weak correlation. Detection method and probe substrates were shown to not be the major cause of the disparity in IC(50)s. However, the enzyme source and composition (HLM versus, rhCYP) caused disparity in IC(50)s. Specifically, the high concentrations of CYP isozymes often used with HLM-based assays produced high probe substrate conversion and test compound metabolism, which should both contribute to artificially higher IC(50)s. Non-specific binding of substrate to higher concentration proteins and lipids in the HLM-based assays should also contribute to higher IC(50)s. The modified double cocktail assay was found to overcome limitations of the other two assays. It uses an rhCYP isozymes mix, drug-probe substrate mix, low protein concentration, and LC-MS detection. The double cocktail assay is sensitive, selective, and high throughout for use in drug discovery to provide an early alert to potential toxicity with regard to drug-drug interaction, prioritize chemical series, and guide structural modification to circumvent CYP450 inhibition.

  3. Screening and Identification of Mitragynine and 7-Hydroxymitragynine in Human Urine by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Hanzhuo Fu


    Full Text Available Kratom is a tree planted in Southeast Asia, including Thailand, Malaysia, Myanmar (Burma and elsewhere in the region. A long history of usage and abuse of kratom has led to the classification of kratom as a controlled substance in its native Thailand and other Southeast Asian countries. However, kratom is not controlled in the United States, and the wide availability of kratom on the Internet and in the streets has led to its emergence as an herbal drug of misuse. With the increasing popularity of kratom, efficient protocols are needed to detect kratom use. In this study, a rapid method for the analysis of kratom compounds, mitragynine and 7-hydroxymitragynine, in human urine has been developed and validated using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS. The chromatographic system employed a 2.6-μm 100 mm × 2.1 mm phenyl-hexyl analytical column and gradient elution with a 0.4-mL/min flow rate of water and acetonitrile as mobile phases. A triple quadrupole mass spectrometer was used as the detector for data acquisition. The analyst was the quantification software. The established method demonstrated linearity of >0.99 for both analytes, and low detection limits were obtained down to 0.002581 ng/mL for mitragynine and 0.06910 ng/mL for 7-hydroxymitragynine. The validated method has been utilized for clinical analysis of urine for the purpose of mitragynine and 7-hydroxymitragynine detection.

  4. Degradation of pesticides with RSDL®(reactive skin decontamination lotion kit) lotion: LC-MS investigation. (United States)

    Fentabil, Messele; Gebremedhin, Mulu; Purdon, J Garfield; Cochrane, Laura; Goldman, Virginia Streusand


    This study examined the degradation of organophosphate (OP) and carbamate pesticides using RSDL ® (Reactive Skin Decontamination Lotion Kit) lotion. Degradation occurs from a nucleophilic substitution (SN) reaction between an ingredient in the RSDL lotion, potassium 2,3-butanedione monoximate (KBDO), with susceptible sites in the pesticides. Evaluation at several molar ratios of KBDO:test articles using liquid chromatography-mass spectrometry (LC-MS) techniques was performed. The OP test articles, parathion, paraoxon, parathion-methyl, paraoxon-methyl and chlorpyrifos were effectively degraded at molar ratios of four and above in less than 6min contact time. Malathion and malaoxon were similarly converted to inactive by-products at molar ratios as low as two in less than 4min. A minimum molar ratio of nine was found to be effective against the carbamate pesticide carbofuran. In the case of aldicarb, complete destruction was achieved at a molar ratio of fifteen and a reaction time of one hour. It is important to note that these studies are based on a direct liquid phase RSDL lotion reaction with the toxic chemicals without the added physical removal decontamination efficacy component provided by the sponge component of the RSDL kit. The RSDL kit is intended to be used to remove or neutralize chemical warfare agents (CWA) and T-2 toxin from the skin. In actual use, the majority of the CWA decontamination occurs through the combined action of the sponge in both removing the chemical from the skin, and in rapidly mixing the chemicals at a high molar ratio of KBDO:CWA within the pores of the sponge to enhance rapid neutralization of the chemical. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Quantitative monitoring of corticosteroids in cosmetic products manufactured in Korea using LC-MS/MS. (United States)

    Nam, Yun Sik; Kwon, Il Keun; Lee, Yeonhee; Lee, Kang-Bong


    Some cosmetic products manufactured in Korea for the treatment of eczema, seborrhea and psoriasis have been suspected to contain anti-inflammatory corticosteroids such as prednisolone, hydrocortisone, betamethasone, dexamethasone and triamcinolone acetonide without these ingredients being indicated on the label. Due to their severe side effects such as permenent skin atopy, these corticosteroids have to be monitored in cosmetic products from a forensic point of view. Many cosmetic product samples (N=65) have been collected from both local and online markets in Korea. The corticosteroid content of these samples was analyzed by LC-MS/MS with diagnostic ions (m/z). Linearity was studied with 0.1-10 μg/mL range in all corticosteroids. Good correlation coefficients (r(2)≥0.997) were found and the limits of quantification were 4.68-7.97 ng/mL for each of the corticosteroids. At three different concentrations spanning the linear dynamic ranges, mean recoveries were 97.2-113.5%and precisions (RSD) for intra-day and inter-day analysis were less than 8.9%. Also, accuracy (Bias %) was less than 11.8%. The results showed that between 0.76-0.94 μg/g levels of prednisolone were detected in four cosmetic products and triamcinolone acetonidewas detected with a concentration in the range of 11.5-272 μg/g in nine samples. This fact reveals that some manufacturers have arbitrarily added these corticosteroids in their cosmetic products without indicating them on the label. Thus, these cosmetic products have to be monitored and if proven illegal preparations removed from the market. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  6. Identification of carbonylated lipids from different phospholipid classes by shotgun and LC-MS lipidomics. (United States)

    Ni, Zhixu; Milic, Ivana; Fedorova, Maria


    Oxidized lipids play a significant role in the pathogenesis of numerous oxidative stress-related human disorders, such as atherosclerosis, obesity, inflammation, and autoimmune diseases. Lipid peroxidation, induced by reactive oxygen and nitrogen species, yields a high variety of modified lipids. Among them, carbonylated lipid peroxidation products (oxoLPP), formed by oxidation of the fatty acid moiety yielding aldehydes or ketones (carbonyl groups), are electrophilic compounds that are able to modify nucleophilic substrates like proteins, nucleic acid, and aminophospholipids. Some carbonylated phosphatidylcholines possess even pro-inflammatory activities. However, little is known about oxoLPP derived from other phospholipid (PL) classes. Here, we present a new analytical strategy based on the mass spectrometry (MS) of PL-oxoLPP derivatized with 7-(diethylamino)coumarin-3-carbohydrazide (CHH). Shotgun MS revealed many oxoLPP derived from in vitro oxidized glycerophosphatidylglycerols (PG, 31), glycerophosphatidylcholine (PC, 23), glycerophosphatidylethanolamine (PE, 34), glycerophosphatidylserines (PS, 7), glycerophosphatidic acids (PA, 17), and phosphatidylinositiolphosphates (PIP, 6) vesicles. This data were used to optimize LipidXplorer-assisted identification, and a python-based post-processing script was developed to increase both throughput and accuracy. When applied to full lipid extracts from rat primary cardiomyocytes treated with peroxynitrite donor SIN-1, ten PL-bound oxoLPP were unambiguously identified by LC-MS, including two PC-, two PE-, one PG-, two PS-, and three PA-derived species. Some of the well-known carbonylated PC were detected, while most PL-oxoLPP were shown for the first time.

  7. Identification of p90 Ribosomal S6 Kinase 2 as a Novel Host Protein in HBx Augmenting HBV Replication by iTRAQ-Based Quantitative Comparative Proteomics. (United States)

    Yan, Li-Bo; Yu, You-Jia; Zhang, Qing-Bo; Tang, Xiao-Qiong; Bai, Lang; Huang, FeiJun; Tang, Hong


    The aim of this study was to screen for novel host proteins that play a role in HBx augmenting Hepatitis B virus (HBV) replication. Three HepG2 cell lines stably harboring different functional domains of HBx (HBx, HBx-Cm6, and HBx-Cm16) were cultured. ITRAQ technology integrated with LC-MS/MS analysis was applied to identify the proteome differences among these three cell lines. In brief, a total of 70 different proteins were identified among HepG2-HBx, HepG2-HBx-Cm6, and HepG2-HBx-Cm16 by double repetition. Several differentially expressed proteins, including p90 ribosomal S6 kinase 2 (RSK2), were further validated. RSK2 was expressed at higher levels in HepG2-HBx and HepG2-HBx-Cm6 compared with HepG2-HBx-Cm16. Furthermore, levels of HBV replication intermediates were decreased after silencing RSK2 in HepG2.2.15. An HBx-minus HBV mutant genome led to decreased levels of HBV replication intermediates and these decreases were restored to levels similar to wild-type HBV by transient ectopic expression of HBx. After silencing RSK2 expression, the levels of HBV replication intermediates synthesized from the HBx-minus HBV mutant genome were not restored to levels that were observed with wild-type HBV by transient HBx expression. Based on iTRAQ quantitative comparative proteomics, RSK2 was identified as a novel host protein that plays a role in HBx augmenting HBV replication. © 2018 The Authors. Proteomics - Clinical Application Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. GridMass: a fast two-dimensional feature detection method for LC/MS. (United States)

    Treviño, Victor; Yañez-Garza, Irma-Luz; Rodriguez-López, Carlos E; Urrea-López, Rafael; Garza-Rodriguez, Maria-Lourdes; Barrera-Saldaña, Hugo-Alberto; Tamez-Peña, José G; Winkler, Robert; Díaz de-la-Garza, Rocío-Isabel


    One of the initial and critical procedures for the analysis of metabolomics data using liquid chromatography and mass spectrometry is feature detection. Feature detection is the process to detect boundaries of the mass surface from raw data. It consists of detected abundances arranged in a two-dimensional (2D) matrix of mass/charge and elution time. MZmine 2 is one of the leading software environments that provide a full analysis pipeline for these data. However, the feature detection algorithms provided in MZmine 2 are based mainly on the analysis of one-dimension at a time. We propose GridMass, an efficient algorithm for 2D feature detection. The algorithm is based on landing probes across the chromatographic space that are moved to find local maxima providing accurate boundary estimations. We tested GridMass on a controlled marker experiment, on plasma samples, on plant fruits, and in a proteome sample. Compared with other algorithms, GridMass is faster and may achieve comparable or better sensitivity and specificity. As a proof of concept, GridMass has been implemented in Java under the MZmine 2 environment and is available at and MASSyPup. It has also been submitted to the MZmine 2 developing community. Copyright © 2015 John Wiley & Sons, Ltd.

  9. Core proteome of the minimal cell: comparative proteomics of three mollicute species.

    Directory of Open Access Journals (Sweden)

    Gleb Y Fisunov

    Full Text Available Mollicutes (mycoplasmas have been recognized as highly evolved prokaryotes with an extremely small genome size and very limited coding capacity. Thus, they may serve as a model of a 'minimal cell': a cell with the lowest possible number of genes yet capable of autonomous self-replication. We present the results of a comparative analysis of proteomes of three mycoplasma species: A. laidlawii, M. gallisepticum, and M. mobile. The core proteome components found in the three mycoplasma species are involved in fundamental cellular processes which are necessary for the free living of cells. They include replication, transcription, translation, and minimal metabolism. The members of the proteome core seem to be tightly interconnected with a number of interactions forming core interactome whether or not additional species-specific proteins are located on the periphery. We also obtained a genome core of the respective organisms and compared it with the proteome core. It was found that the genome core encodes 73 more proteins than the proteome core. Apart of proteins which may not be identified due to technical limitations, there are 24 proteins that seem to not be expressed under the optimal conditions.

  10. [Functional analysis of cancer-derived immunoglobulin G whole molecule-interacting proteins identified by LC-MS/MS]. (United States)

    Wang, Ju-Ping; Chen, Han-Ying; Peng, Hui


    To identify cancer-derived immunoglobulin G (IgG) whole molecule-interacting proteins to provide important clues for studying IgG biological functions. HeLa cell lysate was immunoprecipitated with rabbit antihuman IgG whole molecule antibody and normal rabbit IgG. The immunocomplex underwent sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and was detected with silver staining. Three prominently enhanced bands were subjected to protein identification with liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the MS data were analyzed with Swiss-Prot database. Cancer-derived IgG whole molecule-interacting proteins were screened and functionally annotated. We identified 6 potential cancer-derived IgG whole molecule-interacting proteins with co-immunoprecipitation combined with LC-MS/MS, which provides valuable clues for studying the function of cancer-derived IgG.

  11. Post-acquisition data mining techniques for LC-MS/MS-acquired data in drug metabolite identification. (United States)

    Dhurjad, Pooja Sukhdev; Marothu, Vamsi Krishna; Rathod, Rajeshwari


    Metabolite identification is a crucial part of the drug discovery process. LC-MS/MS-based metabolite identification has gained widespread use, but the data acquired by the LC-MS/MS instrument is complex, and thus the interpretation of data becomes troublesome. Fortunately, advancements in data mining techniques have simplified the process of data interpretation with improved mass accuracy and provide a potentially selective, sensitive, accurate and comprehensive way for metabolite identification. In this review, we have discussed the targeted (extracted ion chromatogram, mass defect filter, product ion filter, neutral loss filter and isotope pattern filter) and untargeted (control sample comparison, background subtraction and metabolomic approaches) post-acquisition data mining techniques, which facilitate the drug metabolite identification. We have also discussed the importance of integrated data mining strategy.

  12. Quantitation of Buprenorphine, Norbuprenorphine, Buprenorphine Glucuronide, Norbuprenorphine Glucuronide, and Naloxone in Urine by LC-MS/MS. (United States)

    Marin, Stephanie J; McMillin, Gwendolyn A


    Buprenorphine is an opioid drug that has been used to treat opioid dependence on an outpatient basis, and is also prescribed for managing moderate to severe pain. Some formulations of buprenorphine also contain naloxone to discourage misuse. The major metabolite of buprenorphine is norbuprenorphine. Both compounds are pharmacologically active and both are extensively metabolized to their glucuronide conjugates, which are also active metabolites. Direct quantitation of the glucuronide conjugates in conjunction with free buprenorphine, norbuprenorphine, and naloxone in urine can distinguish compliance with prescribed therapy from specimen adulteration intended to mimic compliance with prescribed buprenorphine. This chapter quantitates buprenorphine, norbuprenorphine, their glucuronide conjugates and naloxone directly in urine by liquid chromatography tandem mass spectrometry (LC-MS/MS). Urine is pretreated with formic acid and undergoes solid phase extraction (SPE) prior to analysis by LC-MS/MS.

  13. Analysis of raw materials, intermediates and subsidiary colours in Food Yellow No. 5 (Sunset Yellow FCF) by LC/MS. (United States)

    Yamada, M; Kawahara, A; Nakamura, M; Nakazawa, H


    Raw materials, intermediates and subsidiary colours in Food Yellow No. 5 (Sunset Yellow FCF) were determined using liquid chromatography/mass spectrometry (LC/MS) with electrospray ionization. A gradient consisting of acetonitrile and 0.04% aqueous ammonium carbonate solution was used for the HPLC mobile phase. Quasi-molecular ions of impurities were used as monitor ions. It was necessary to use fragment ions of the sodium salts of 6-hydroxy-5-phenylazo-2-naphthalenesulphonic acid (SS-AN) and 4-(2-hydroxy-1-naphthylazo) benzenesulphonic acid (2N-SA) as monitor ions because the compounds are not resolved by chromatography and have the same molecular weight. Fifteen samples of commercial Sunset Yellow FCF were examined. The results obtained by UV-Vis spectroscopy were in good agreement with the results of LC/MS analyses. The detection limits of the impurities in Sunset Yellow FCF ranged from 0.01 to 0.1%.

  14. Quantitative analysis of pharmacokinetic study samples by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). (United States)

    Mück, W


    The basis of all pharmacokinetic evaluations are powerful assays to quantify drugs and/or metabolites in biological matrices using modern sensitive instrumental analytical techniques, such as capillary gas chromatography and high-performance liquid chromatography (HPLC). Being both specific and universal, mass spectrometry (MS) is an ideal chromatographic detector. Due to recent exciting achievements in the interfacing of liquid chromatography (LC) and MS, LC-MS, like the successfully preceding hyphenated technique gas chromatography-mass spectrometry (GC-MS), has now become a valuable technique in the analyst's toolbox. The key features of LC-MS are explained and four examples demonstrating its potential for highly specific and sensitive routine drug assays with the option of high sample throughput in pharmacokinetic investigations are presented.

  15. LC-MS/MS based identification of piperine production by endophytic Mycosphaerella sp. PF13 from Piper nigrum. (United States)

    Chithra, S; Jasim, B; Anisha, C; Mathew, Jyothis; Radhakrishnan, E K


    Piper nigrum is very remarkable for its medicinal properties due to the presence of metabolites like piperine. Emerging understanding on the biosynthetic potential of endophytic fungi suggests the possibility to have piperine producing fungi in P. nigrum. In the current study, endophytic fungi isolated from P. nigrum were screened for the presence of piperine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This resulted in the identification of a Mycosphaerella sp. with the ability to produce piperine extracellularly. The biosynthesis of piperine (C17H19NO3) by the endophytic fungal isolate was confirmed by the presence of m/z 286.1 (M + H(+)) in the LC-MS/MS analysis using positive mode ionization. This was further supported by the presence of specific fragment ions with masses 135, 143, 171 and 201 formed due to the fragmentation of piperine present in the fungal extract.

  16. Development of an LC-MS/MS method for the determination of pesticides and patulin in apples

    DEFF Research Database (Denmark)

    Christensen, Hanne Bjerre; Poulsen, Mette Erecius; Rasmussen, Peter Have


    A method for the simultaneous determination of 33 pesticides or degradation products together with patulin in apples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method involved homogenization of the apples, extraction with ammonium acetate-acetic acid solution...... in methanol-water by ultrasonication, filtration, and determination by LC-MS/MS. The repeatability and within-laboratory reproducibility for the three spiking levels 0.02, 0.04 and 0.2 mg kg(-1) were between 4% and 35%. In general, the repeatability and reproducibility were about 10-20%. The limits...... of quantification (LOQs) were between 0.01 and 0.14 mg kg(-1). The method was used on incurred samples from parts of the ISAFRUIT project financed by the European Commission under the 6th Framework Programme. Samples were analysed at four different stages: after harvest, after storage (controlled), after a water...

  17. UV filters analyzed by isotope diluted TurboFlow-LC-MS/MS in urine from Danish children and adolescents

    DEFF Research Database (Denmark)

    Frederiksen, Hanne; Nielsen, Ole; Skakkebaek, Niels E


    between first morning and 24h urine levels of BP-3, BP-1 and 4-HBP from the same day was observed. CONCLUSION: Our project on UV filters analyzed by a new robust and sensitive LC-MS/MS method in Danish children and adolescents showed that almost all individuals were exposed to UV filters. Sun protection...... and adolescents (6-21 yrs). All 387 samples were collected during the autumn (Nov. 2007) and were analyzed by a new on-line TurboFlow-LC-MS/MS method developed for simultaneous biomonitoring of these nine UV filters in urine. RESULTS: BP-3 and BP-1 were detected in more than 80% of the 24h samples and were...

  18. A simple LC-MS/MS method for determination of deferasirox in human plasma: Troubleshooting of interference from ferric ion in method development and its application. (United States)

    Li, Tengfei; Cui, Zhimin; Wang, Yan; Yang, Wen; Li, Duo; Song, QinXin; Sun, Luning; Ding, Li


    As an orally active iron chelator, deferasirox forms its ion complexes in the prepared plasma samples and LC-MS mobile phase where ferric ion exists, and then comparing with the nominal concentration level, a lower detected concentration level of deferasirox would be obtained after LC-MS analysis, if no proper treatment was adopted. Meanwhile, the phenomenon would be observed that multiple repeat injections of the same deferasirox plasma sample in the same tube would show the lower and lower detected concentration levels of deferasirox, which caused by more and more ferric ions from the injection needle dissolved in the sample solution as multiple repeated injections. The addition of a proper concentration of EDTA in the mobile phase and the sample will competitively inhibit deferasirox from complexing with ferric ion, and prevent the decrease of deferasirox concentration. In this paper, an LC-MS/MS method was developed and validated for the determination of deferasirox in human plasma. To achieve the protein precipitation, the analytes were extracted from aliquots of 200 μL human plasma with acetonitrile. Chromatographic separation was performed on an ODS-C18 column with the mobile phase consisted of methanol and 0.1% formic acid containing 0.04 mM ethylenediamine tetraacetate dihydrate (EDTA) (80:20, v/v) at a flow rate of 0.5 mL/min. Deferasirox and the internal standard (IS, mifepristone) were detected using electrospray ionization in positive ion multiple reaction monitoring mode by monitoring the precursor-to-product ion transitions m/z 374.2 → 108.1 for deferasirox and m/z 430.1 → 372.2 for the IS. The method exhibited good linearity over the concentration range of 0.04-40 μg/mL for deferasirox. The method was successfully applied to a pharmacokinetic study in 10 Chinese healthy volunteers after oral administration of deferasirox. Copyright © 2018 Elsevier B.V. All rights reserved.

  19. Comparative proteomics and activity of a green sulfur bacterium across the water column of Lake Cadagno, Switzerland

    DEFF Research Database (Denmark)

    Habicht, Kirsten Silvia; Miller, Mette; Cox, Raymond Pickett


    the bacteria survive in the dark. Although metagenomic data are not available for Lake Cadagno, proteome analysis was possible based on the completely sequenced genome of an isolated strain of Chl. clathratiforme. Using LC-MS/MS we identified 1321 Chl. clathratiforme proteins in Lake Cadagno and quantitatively...... participating in nitrogen and sulfur metabolism were twofold less abundant in the dark. From the proteome analysis we were able to show that Chl. clathratiforme in the photic zone contains enzymes for fixation of N2 and the complete oxidation of sulfide to sulfate while these processes are probably not active......Primary production in the meromictic Lake Cadagno, Switzerland, is dominated by anoxygenic photosynthesis. The green sulfur bacterium Chlorobium clathratiforme is the dominant phototrophic organism in the lake, comprising more than half of the bacterial population, and its biomass increases 3...

  20. Bisphenol A release from an orthodontic resin composite: A GC/MS and LC/MS study. (United States)

    Deviot, Marc; Lachaise, Isabelle; Högg, Christof; Durner, Jürgen; Reichl, Franz-Xaver; Attal, Jean-Pierre; Dursun, Elisabeth


    First, to analyse the in vitro release of BPA and Bis-GMA from an orthodontic resin composite (Transbond XT, 3M Unitek), stored in various conditions, by gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS); then to extrapolate the data to the clinical situation. Secondly, to explore the thermal stability of Bis-GMA. Cylinders of resin composite were prepared and stored according to 3 different protocols: (1) they were light-cured 20s, then placed in artificial saliva; (2) they were light-cured 2s, then placed in acetonitrile; (3) they were light-cured 2s, then placed in methanol. For each group, BPA and Bis-GMA release were determined with GC/MS and/or LC/MS at least after one week. Besides, 120 brackets (10 of each type) were bonded over metal teeth, then debonded, and the weight and the surface of resin composite residues were measured. BPA and Bis-GMA release of adhesive residues were extrapolated from the data obtained with the cylinders. Besides, BPA release from a heated Bis-GMA solution was measured. With GC/MC, BPA was detected in all samples. With LC/MS, BPA was detected only from samples immersed in MeOH; Bis-GMA was detected, in varying amount according to the extraction media and the light-curing time. BPA was found after heating of the Bis-GMA solution. Contamination risk and the heat applied in GC/MS may overestimate the BPA release from resin composite. Based on the LC/MS results, the risk of BPA release after orthodontic bonding would be more than 42000 times lower than the TDI for a 30-kg child. Copyright © 2017 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

  1. Determination of isosteviol by LC-MS/MS and its application for evaluation of pharmacokinetics of isosteviol in rat

    Directory of Open Access Journals (Sweden)

    Bazargan M.


    Full Text Available Isosteviol has been found to have potential preventive or therapeutic effects against hypertension, ischemia reperfusion injury, diabetes and cancer, but little is known about the pharmacokinetics (PK of the compound. The aim of this study was to develop a liquid chromatographic-tandem mass spectrometric (LC-MS/MS method for determination of isosteviol in rat plasma and to assess in a preliminary manner the PK of isosteviol after intravenous bolus injection.Ions of analytes were generated using electro-spray ionization and detected in the positive-ion mode in LC-MS/MS. Multiple reaction monitoring was performed, using the precursor product ion combination for isosteviol m/z 319.4→273.4. Progesterone was used as an internal standard. Nitrogen was used as the nebulising gas and unit resolution was set for Q1 and Q3. Isosteviol solution was injected through the penile vein of rats at a dose of 8 mg/kg. Blood samples were collected from a jugular vein cannula. The PK parameters were calculated using a two - compartment PK model.The LC-MS/MS assay for isosteviol in rat plasma was linear over the range of 0.5-80 μg/ml. The terminal half life of isosteviol (t 1/2 was 406 ± 31.7 min and clearance (CL was 2.9 ± 0.3 ml/min/kg. A sensitive LC-MS/MS assay for isosteviol in plasma has been successfully established and used in a preliminary PK evaluation of isosteviol in rats.

  2. Effect of Genetics, Environment, and Phenotype on the Metabolome of Maize Hybrids Using GC/MS and LC/MS. (United States)

    Tang, Weijuan; Hazebroek, Jan; Zhong, Cathy; Harp, Teresa; Vlahakis, Chris; Baumhover, Brian; Asiago, Vincent


    We evaluated the variability of metabolites in various maize hybrids due to the effect of environment, genotype, phenotype as well as the interaction of the first two factors. We analyzed 480 forage and the same number of grain samples from 21 genetically diverse non-GM Pioneer brand maize hybrids, including some with drought tolerance and viral resistance phenotypes, grown at eight North American locations. As complementary platforms, both GC/MS and LC/MS were utilized to detect a wide diversity of metabolites. GC/MS revealed 166 and 137 metabolites in forage and grain samples, respectively, while LC/MS captured 1341 and 635 metabolites in forage and grain samples, respectively. Univariate and multivariate analyses were utilized to investigate the response of the maize metabolome to the environment, genotype, phenotype, and their interaction. Based on combined percentages from GC/MS and LC/MS datasets, the environment affected 36% to 84% of forage metabolites, while less than 7% were affected by genotype. The environment affected 12% to 90% of grain metabolites, whereas less than 27% were affected by genotype. Less than 10% and 11% of the metabolites were affected by phenotype in forage and grain, respectively. Unsupervised PCA and HCA analyses revealed similar trends, i.e., environmental effect was much stronger than genotype or phenotype effects. On the basis of comparisons of disease tolerant and disease susceptible hybrids, neither forage nor grain samples originating from different locations showed obvious phenotype effects. Our findings demonstrate that the combination of GC/MS and LC/MS based metabolite profiling followed by broad statistical analysis is an effective approach to identify the relative impact of environmental, genetic and phenotypic effects on the forage and grain composition of maize hybrids.

  3. LIQUID: an-open source software for identifying lipids in LC-MS/MS-based lipidomics data

    Energy Technology Data Exchange (ETDEWEB)

    Kyle, Jennifer E.; Crowell, Kevin L.; Casey, Cameron P.; Fujimoto, Grant M.; Kim, Sangtae; Dautel, Sydney E.; Smith, Richard D.; Payne, Samuel H.; Metz, Thomas O.


    We introduce an open-source software, LIQUID, for semi-automated processing and visualization of LC-MS/MS based lipidomics data. LIQUID provides users with the capability to process high throughput data and contains a customizable target library and scoring model per project needs. The graphical user interface provides visualization of multiple lines of spectral evidence for each lipid identification, allowing rapid examination of data for making confident identifications of lipid molecular species.


    Directory of Open Access Journals (Sweden)

    Khairan Khairan


    Full Text Available Semisynthesis of D6,7-Anhydroerythromycin-A was done by biomodification technique by addition of 0.2% INH into a culture fermentation of Saccharopolyspora erythraea ATCC 11635 in medium Hutchinson. The aim of this research is to studies of fragmentation pattern from new matabolite of D6,7-Anhydroerythromycin-A by Liquid Chromatography-Mass Spectroscopy (LC-MS and the ionization of mass spectroscopy is use by ESI (Electrospray Ionization pattern. The FT-IR spectrometric analyzes showed a stretching vibration of C=C conjugated group at wave number 1602.7 cm-1. This C=C conjugated vibration indicated the existence of double bond between C6 and C7 (D6,7, this confirmed that isolate contained D6,7-Anhydroerythromycin-A (the possibility of D6,7 was positive. For complementation, a LC-MS (Liquid Chromatography-Mass Spectroscopy analyzes using ESI-MS (Electrospray Ionization-Mass Spectroscopy ionization pattern was conducted to the isolate which resulted Quassimolecular ions [M+H]+ of D7,8- and D6,7-Anhydroerythromycin-A. LC-MS spectrogram of the isolate, which gave two peaks of m/z 732.2460 and m/z 716.2522, confirmed that the m/z 732.2460 possibly was D7,8-Anhydroerythromycin-A, while the m/z 716.2502 and m/z 715.2522 possibly were D6,7-Anhydroerythromycin-A.   Keywords: isoniazid, enoyl reduction, D6,7-Anhidroeritromisin-A, fragmentation, LC-MS.

  5. IMAC fractionation in combination with LC-MS reveals H2B and NIF-1 peptides as potential bladder cancer biomarkers. (United States)

    Frantzi, Maria; Zoidakis, Jerome; Papadopoulos, Theofilos; Zürbig, Petra; Katafigiotis, Ioannis; Stravodimos, Konstantinos; Lazaris, Andreas; Giannopoulou, Ioanna; Ploumidis, Achilles; Mischak, Harald; Mullen, William; Vlahou, Antonia


    Improvement in bladder cancer (BC) management requires more effective diagnosis and prognosis of disease recurrence and progression. Urinary biomarkers attract special interest because of the noninvasive means of urine collection. Proteomic analysis of urine entails the adoption of a fractionation methodology to reduce sample complexity. In this study, we applied immobilized metal affinity chromatography in combination with high-resolution LC-MS/MS for the discovery of native urinary peptides potentially associated with BC aggressiveness. This approach was employed toward urine samples from patients with invasive BC, noninvasive BC, and benign urogenital diseases. A total of 1845 peptides were identified, corresponding to a total of 638 precursor proteins. Specific enrichment for proteins involved in nucleosome assembly and for zinc-finger transcription factors was observed. The differential expression of two candidate biomarkers, histone H2B and NIF-1 (zinc finger 335) in BC, was verified in independent sets of urine samples by ELISA and by immunohistochemical analysis of BC tissue. The results collectively support changes in the expression of both of these proteins with tumor progression, suggesting their potential role as markers for discriminating BC stages. In addition, the data indicate a possible involvement of NIF-1 in BC progression, likely as a suppressor and through interactions with Sox9 and HoxA1.

  6. Preparation of open tubular capillary columns by in situ ring-opening polymerization and their applications in cLC-MS/MS analysis of tryptic digest. (United States)

    Wang, Hongwei; Yao, Yating; Li, Ya; Ma, Shujuan; Peng, Xiaojun; Ou, Junjie; Ye, Mingliang


    An open tubular (OT) column (25 μm i.d.) was prepared by in situ ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxanes (POSS-epoxy) with 4-aminophenyl disulfide (APDS) in a binary porogenic system of ethanol/H 2 O. It was found that porogenic composition played an important role in the formation of OT stationary phases. The ratio of ethanol/H 2 O at 6/1 (v/v) would lead to the fabrication of hybrid monoliths, while the ratio of ethanol/H 2 O at 13/1 (v/v) would result in the synthesis of OT phases. In addition, the effects of precursor content and reaction duration on the thickness of OT stationary phases were investigated. Either lower precursor content or shorter reaction duration would produce thinner layer of OT column. The repeatability of OT columns was evaluated through relative standard deviation (RSD%) with benzene as the analyte. The run-to-run, column-to-column and batch-to-batch repeatabilities were 1.7%, 4.8% and 5.6%, respectively, exhibiting satisfactory repeatability of the OT column. Then tryptic digest of mouse liver proteins was used to evaluate the performance of the resulting OT columns (25 μm i.d. × 2.5 m in length) by cLC-MS/MS analysis, demonstrating their potential in proteome analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Improved LC-MS/MS of Heparan Sulfate Oligosaccharides via Chip-based Pulsed Make-up Flow (United States)

    Huang, Yu; Shi, Xiaofeng; Yu, Xiang; Leymarie, Nancy; Staples, Gregory O.; Yin, Hongfeng; Killeen, Kevin; Zaia, Joseph


    Microfluidic chip-based hydrophilic interaction chromatography (HILIC) is a useful separation system for liquid chromatography-mass spectrometry (LC-MS) in compositional profiling of heparan sulfate (HS) oligosaccharides; however, ions observed using HILIC LC-MS are low in charge. Tandem MS of HS oligosaccharide ions with low charge results in undesirable losses of SO3 from precursor ions during collision induced dissociation. One solution is to add metal cations to stabilize sulfate groups. Another is to add a non-volatile, polar compound such as sulfolane, a molecule known to supercharge proteins, to produce a similar effect for oligosaccharides. We demonstrate use of a novel pulsed make-up flow (MUF) HPLC-chip. The chip enables controlled application of additives during specified chromatographic time windows and thus minimizes the extent to which non-volatile additives build up in the ion source. The pulsed MUF system was applied to LC-MS/MS of HS oligosaccharides. Metal cations and sulfolane were tested as additives. The most promising results were obtained for sulfolane, for which supercharging of the oligosaccharide ions increased their signal strengths relative to controls. Tandem MS of these supercharged precursor ions showed decreased abundances of product ions from sulfate losses yet more abundant product ions from backbone cleavages. PMID:21923145

  8. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) based bioavailability determination of the major classes of phytochemicals. (United States)

    Stylos, Evgenios; Chatziathanasiadou, Maria V; Syriopoulou, Aggeliki; Tzakos, Andreas G


    Natural products derived from plants have served as an inexhausted source for drug discovery and drug development. They have been evolutionary amplified with drug-like properties and have already illustrated immense therapeutic potential over an array of different diseases. However, their incorporation in the drug discovery pipeline has been diminished the last two decades. This was probably due to barriers related to their inherent difficulties to be integrated in high-throughput screening assays as also their largely unexplored bioavailability. Analytical procedures have come into the spotlight, a result of the continuous development of the instrumentation's capabilities as far as detection and separation is concerned. Integral part of this technological evolution is LC-MS instrumentation and its extended use for the determination of various compounds. The fact that it provides extra sensitivity, specificity and good separation in complex samples, makes LC-MS/MS the ultimate tool in the determination of many types of chemical compounds, such as phytochemicals. Herein, we focus on the achievements of the last five years in quantitative analysis of the major classes of phytochemicals (flavonoids, alkaloids, terpenes, glycosides and saponins) in plasma, through LC-MS/MS, as also their bioavailability. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. An overview of sample preparation procedures for LC-MS multiclass antibiotic determination in environmental and food samples. (United States)

    Moreno-Bondi, María Cruz; Marazuela, María Dolores; Herranz, Sonia; Rodriguez, Erika


    Antibiotics are a class of pharmaceuticals that are of great interest due to the large volumes of these substances that are consumed in both human and veterinary medicine, and due to their status as the agents responsible for bacterial resistance. They can be present in foodstuffs and in environmental samples as multicomponent chemical mixtures that exhibit a wide range of mechanisms of action. Moreover, they can be transformed into different metabolites by the action of microorganisms, as well as by other physical or chemical means, resulting in mixtures with higher ecotoxicities and risks to human health than those of the individual compounds. Therefore, there is growing interest in the availability of multiclass methods for the analysis of antimicrobial mixtures in environmental and food samples at very low concentrations. Liquid chromatography (LC) has become the technique of choice for multiclass analysis, especially when coupled to mass spectrometry (LC-MS) and tandem MS (LC-MS(2)). However, due to the complexity of the matrix, in most cases an extraction step for sample clean-up and preconcentration is required before analysis in order to achieve the required sensitivities. This paper reviews the most recent developments and applications of multiclass antimicrobial determination in environmental and food matrices, emphasizing the practical aspects of sample preparation for the simultaneous extraction of antimicrobials from the selected samples. Future trends in the application of LC-MS-based techniques to multiclass antibiotic analysis are also presented.

  10. Simultaneous Determination of Cyclosporine A, Tacrolimus, Sirolimus, and Everolimus in Whole-Blood Samples by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Mustafa Karapirli


    Full Text Available Objectives. Cyclosporine A (CyA, tacrolimus (TRL, sirolimus (SIR, and everolimus (RAD are immunosuppressive drugs frequently used in organ transplantation. Our aim was to confirm a robust sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS method for determination of CyA, TRL, SIR, and RAD in whole-blood samples. Materials and Methods. We used an integrated online solid-phase extraction-LC-MS/MS system and atmospheric pressure ionization tandem mass spectrometry (API-MS/MS in the multiple reaction monitoring (MRM detection mode. CyA, TRL, SIR, and RAD were simultaneously analyzed in whole blood treated with precipitation reagent taken from transplant patients. Results. System performance parameters were suitable for using this method as a high-throughput technique in clinical practice. The high concentration of one analyte in the sample did not affect the concentration of other analytes. Total analytical time was 2.5 min, and retention times of all analytes were shorter than 2 minutes. Conclusion. This LC-MS/MS method can be preferable for therapeutic drug monitoring of these immunosuppressive drugs (CyA, TRL, SRL, and RAD in whole blood. Sample preparation was too short and simple in this method, and it permits robust, rapid, sensitive, selective, and simultaneous determination of these drugs.

  11. Intracellular Drug Uptake-A Comparison of Single Cell Measurements Using ToF-SIMS Imaging and Quantification from Cell Populations with LC/MS/MS. (United States)

    Newman, Carla F; Havelund, Rasmus; Passarelli, Melissa K; Marshall, Peter S; Francis, Ian; West, Andy; Alexander, Morgan R; Gilmore, Ian S; Dollery, Colin T


    ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.

  12. Effects of sample injection amount and time-of-flight mass spectrometric detection dynamic range on metabolome analysis by high-performance chemical isotope labeling LC-MS. (United States)

    Zhou, Ruokun; Li, Liang


    The effect of sample injection amount on metabolome analysis in a chemical isotope labeling (CIL) liquid chromatography-mass spectrometry (LC-MS) platform was investigated. The performance of time-of-flight (TOF) mass spectrometers with and without a high-dynamic-range (HD) detection system was compared in the analysis of (12)C2/(13)C2-dansyl labeled human urine samples. An average of 1635 ± 21 (n = 3) peak pairs or putative metabolites was detected using the HD-TOF-MS, compared to 1429 ± 37 peak pairs from a conventional or non-HD TOF-MS. In both instruments, signal saturation was observed. However, in the HD-TOF-MS, signal saturation was mainly caused by the ionization process, while in the non-HD TOF-MS, it was caused by the detection process. To extend the MS detection range in the non-HD TOF-MS, an automated switching from using (12)C to (13)C-natural abundance peaks for peak ratio calculation when the (12)C peaks are saturated has been implemented in IsoMS, a software tool for processing CIL LC-MS data. This work illustrates that injecting an optimal sample amount is important to maximize the metabolome coverage while avoiding the sample carryover problem often associated with over-injection. A TOF mass spectrometer with an enhanced detection dynamic range can also significantly increase the number of peak pairs detected. In chemical isotope labeling (CIL) LC-MS, relative metabolite quantification is done by measuring the peak ratio of a (13)C2-/(12)C2-labeled peak pair for a given metabolite present in two comparative samples. The dynamic range of peak ratio measurement does not need to be very large, as only subtle changes of metabolite concentrations are encountered in most metabolomic studies where relative metabolome quantification of different groups of samples is performed. However, the absolute concentrations of different metabolites can be very different, requiring a technique to provide a wide detection dynamic range to allow the detection of as

  13. Discrimination of conventional and organic white cabbage from a long-term field trial study using untargeted LC-MS-based metabolomics

    DEFF Research Database (Denmark)

    Mie, Axel; Laursen, Kristian Holst; Åberg, K. Magnus


    The influence of organic and conventional farming practices on the content of single nutrients in plants is disputed in the scientific literature. Here, large-scale untargeted LC-MS-based metabolomics was used to compare the composition of white cabbage from organic and conventional agriculture...... (p = 0.013) imprint in the white cabbage metabolome that is retained between production years. We externally validated this finding by predicting the production system of samples from one year using a classification model built on samples from the other year, with a correct classification in 83...... % of cases. Thus, it was concluded that the investigated conventional and organic management practices have a systematic impact on the metabolome of white cabbage. This emphasizes the potential of untargeted metabolomics for authenticity testing of organic plant products....

  14. Assessment of the occurrence and distribution of pharmaceuticals in a Mediterranean wetland (L'Albufera, Valencia, Spain) by LC-MS/MS. (United States)

    Vazquez-Roig, Pablo; Andreu, Vicente; Onghena, Matthias; Blasco, Cristina; Picó, Yolanda


    The distribution of 17 pharmaceuticals between water and the solid phase (sediments and soils) was studied by utilizing solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two extraction procedures for soils and sediments, prior to the SPE, one based on pressurized liquid extraction (PLE) with hot water and the other on methanol/water ultrasonic extraction, were compared. Absolute recoveries were 71.2-99.3% [relative standard deviation (RSD) MQL) to 35.83 ng g(-1). Among the 17 target compounds, ofloxacin, ciprofloxacin, norfloxacin, trimethoprim, clofibric acid and diclofenac were not detected in soil samples. The average concentrations ranged from less than the MQL for ibuprofen to 34.91 ng g(-1) for tetracycline. These results indicate that pharmaceuticals could survive the wastewater treatment processes, which could lead to their dissemination in water environments.

  15. Photocatalytic degradation of Congo Red by hydrothermally synthesized nanocrystalline TiO2 and identification of degradation products by LC-MS

    International Nuclear Information System (INIS)

    Erdemoglu, Sema; Aksu, Songuel Karaaslan; Sayilkan, Funda; Izgi, Belgin; Asiltuerk, Meltem; Sayilkan, Hikmet; Frimmel, Fritz; Guecer, Seref


    Degradation of Congo Red (CR) dye in aqueous solutions was investigated by means of photocatalysis of TiO 2 which was hydrothermally synthesized at 200 deg. C in 2 h, in anatase phase with 8 nm crystallite size. Efficiency of TiO 2 in photocatalytic degradation under visible irradiation was studied by investigating the effects of amount of TiO 2 , irradiation time, initial CR concentration and pH. It was found that complete decolorization is achieved within 30 min of irradiation. Effects of nitrate and sulphate ions and humic acid on the degradation were also tested. The results were compared with Degussa P-25 TiO 2 at the same degradation conditions. Degradation products were detected using LC-MS technique. The probable pathways for the formation of degradation products were proposed

  16. Building ProteomeTools based on a complete synthetic human proteome (United States)

    Zolg, Daniel P.; Wilhelm, Mathias; Schnatbaum, Karsten; Zerweck, Johannes; Knaute, Tobias; Delanghe, Bernard; Bailey, Derek J.; Gessulat, Siegfried; Ehrlich, Hans-Christian; Weininger, Maximilian; Yu, Peng; Schlegl, Judith; Kramer, Karl; Schmidt, Tobias; Kusebauch, Ulrike; Deutsch, Eric W.; Aebersold, Ruedi; Moritz, Robert L.; Wenschuh, Holger; Moehring, Thomas; Aiche, Stephan; Huhmer, Andreas; Reimer, Ulf; Kuster, Bernhard


    The ProteomeTools project builds molecular and digital tools from the human proteome to facilitate biomedical and life science research. Here, we report the generation and multimodal LC-MS/MS analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products and exemplify the utility of this data. The resource will be extended to >1 million peptides and all data will be shared with the community via ProteomicsDB and proteomeXchange. PMID:28135259

  17. A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Bonaventure Gustavo


    Full Text Available Abstract Background Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices. Results A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA and small polar molecules (e.g., jasmonic acid (JA, salicylic acid (SA containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of Nicotiana attenuata leaf material with previously described analytical methods. Conclusion The method presented was used to detect 16 compounds in leaf extracts of N. attenuata plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the

  18. A rapid and sensitive method for the simultaneous analysis of aliphatic and polar molecules containing free carboxyl groups in plant extracts by LC-MS/MS. (United States)

    Kallenbach, Mario; Baldwin, Ian T; Bonaventure, Gustavo


    Aliphatic molecules containing free carboxyl groups are important intermediates in many metabolic and signalling reactions, however, they accumulate to low levels in tissues and are not efficiently ionized by electrospray ionization (ESI) compared to more polar substances. Quantification of aliphatic molecules becomes therefore difficult when small amounts of tissue are available for analysis. Traditional methods for analysis of these molecules require purification or enrichment steps, which are onerous when multiple samples need to be analyzed. In contrast to aliphatic molecules, more polar substances containing free carboxyl groups such as some phytohormones are efficiently ionized by ESI and suitable for analysis by LC-MS/MS. Thus, the development of a method with which aliphatic and polar molecules -which their unmodified forms differ dramatically in their efficiencies of ionization by ESI- can be simultaneously detected with similar sensitivities would substantially simplify the analysis of complex biological matrices. A simple, rapid, specific and sensitive method for the simultaneous detection and quantification of free aliphatic molecules (e.g., free fatty acids (FFA)) and small polar molecules (e.g., jasmonic acid (JA), salicylic acid (SA)) containing free carboxyl groups by direct derivatization of leaf extracts with Picolinyl reagent followed by LC-MS/MS analysis is presented. The presence of the N atom in the esterified pyridine moiety allowed the efficient ionization of 25 compounds tested irrespective of their chemical structure. The method was validated by comparing the results obtained after analysis of Nicotiana attenuata leaf material with previously described analytical methods. The method presented was used to detect 16 compounds in leaf extracts of N. attenuata plants. Importantly, the method can be adapted based on the specific analytes of interest with the only consideration that the molecules must contain at least one free carboxyl group.

  19. Comparison of the anti-inflammatory active constituents and hepatotoxic pyrrolizidine alkaloids in two Senecio plants and their preparations by LC-UV and LC-MS. (United States)

    Chen, Pinghong; Wang, Yi; Chen, Lulin; Jiang, Wei; Niu, Yan; Shao, Qing; Gao, Lu; Zhao, Quancheng; Yan, Licheng; Wang, Shufang


    Two Senecio plants, Senecio cannabifolius Less. and its variety S. cannabifolius Less. var. integrifolius (Kiodz.) Kidam., were both used as the raw material of Feining granule, a traditional Chinese medicine product for treating respiratory diseases. In this study, the chemical profiles of these two plants were investigated and compared by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). A total number of 83 constituents, including 55 organic acids, 11 flavonoids, 4 alkaloids, 3 terpenes and 10 other types of compounds, were characterized. The results indicated that the levels of most flavonoids were higher in S. cannabifolius than in S. cannabifolius var. integrifolius, however, the levels of hepatotoxic pyrrolizidine alkaloids (PAs) were higher in S. cannabifolius var. integrifolius than in S. cannabifolius. Fifteen constituents were evaluated on lipopolysaccharides (LPS) induced RAW 264.7 cells, and eleven of them showed inhibition effect against nitric oxide (NO) production. Finally, the levels of ten major constituents (including seven anti-inflammatory active ones) and two PAs in Feining granule from two Senecio plants were determined and compared by the LC-UV and LC-MS methods, respectively. It was found that one organic acid (homogentisic acid) and two PAs (seneciphylline and senecionine) had higher contents in the preparation of S. cannabifolius var. integrifolius than in that of S. cannabifolius, however, the situations were inverse for the levels of four organic acids and flavonoids (chlorogenic acid, hyperoside, isoquercitrin, and isochlorogenic acid B). Based on the above results, S. cannabifolius might be a better raw material for Feining granule than S. cannabifolius var. integrifolius, because it contained more anti-inflammatory constituents and less hepatotoxic PAs than the latter. However, more pharmacological evaluations should be carried out to support the selection. The results in this study were helpful

  20. Determination of Scopolamine in Human Saliva Using Solid Phase Extraction and LC/MS/MS (United States)

    Wang, Zuwei; Vaksman, Zalman; Boyd, Jason; Putcha, Lakshmi


    Purpose: Scopolamine is the preferred treatment for motion sickness during space flight because of its quick onset of action, short half-life and favorable side-effect profile. The dose administered depends on the mode of administration and usually ranges between 0.1 and 0.8 mg. Such small doses make it difficult to detect concentrations of scopolamine in biological fluids by using conventional HPLC methods. To measure scopolamine in saliva and thereby to evaluate the pharmacokinetics of scopolamine, we developed an LC/MS/MS method using off-line solid phase extraction. Method: Samples (0.5mL) were loaded onto Waters Oasis HLB co-polymer cartridges (10 mg, 1 mL) and eluted with 0.5 mL methanol without evaporation and reconstitution. HPLC separation of the eluted sample was performed using an Agilent Zorbax SB-CN column (50 x 2.1 mm) at a flow rate of 0.2 mL/min for 4 minutes. The mobile phase for separation was 90:10 (v/v) methanol: ammonium acetate (2 mM) in water, pH 5.0 +/- 0.1. Concentrations of scopolamine were determined using a Micromass Quattro Micro(TM) mass spectrometer with electrospray ionization (ESI). ESI mass spectra were acquired in positive ion mode with multiple reaction monitoring for the determination of scopolamine m/z = 304.2 yields 138.1 and internal standard (IS) hyoscyamine m/z = 290.2 yields 124.1. Results: The method is rapid, reproducible, specific and has the following parameters: scopolamine and the IS are eluted at 1.7 and 3.2 min respectively. The linear range is 50-5000 pg/mL for scopolamine in saliva with correlation coefficients > 0.99 with a CV < 0.5 %. The intra-day and inter-day CVs are < 15 % for quality control samples with concentrations of 75, 300, 750 and 3000 pg/mL of scopolamine in human saliva. Conclusion: Solid phase extraction allows more rapid sample preparation and greater precision than liquid extraction. Furthermore, we increased the sensitivity and specificity by adjusting the LC mobile phase and using an MS

  1. Preservation of urine free catecholamines and their free O-methylated metabolites with citric acid as an alternative to hydrochloric acid for LC-MS/MS-based analyses. (United States)

    Peitzsch, Mirko; Pelzel, Daniela; Lattke, Peter; Siegert, Gabriele; Eisenhofer, Graeme


    Measurements of urinary fractionated metadrenalines provide a useful screening test to diagnose phaeochromocytoma. Stability of these compounds and their parent catecholamines during and after urine collection is crucial to ensure accuracy of the measurements. Stabilisation with hydrochloric acid (HCl) can promote deconjugation of sulphate-conjugated metadrenalines, indicating a need for alternative preservatives. Urine samples with an intrinsically acidic or alkaline pH (5.5-6.9 or 7.1-8.7, respectively) were used to assess stability of free catecholamines and their free O-methylated metabolites over 7 days of room temperature storage. Stabilisation with HCl was compared with ethylenediaminetetraacetic acid/metabisulphite and monobasic citric acid. Catecholamines and metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Free catecholamines and their O-methylated metabolites were stable in acidic urine samples over 7 days of room temperature storage, independent of the presence or absence of any stabilisation method. In contrast, free catecholamines, but not the free O-methylated metabolites, showed rapid degradation within 24 h and continuing degradation over 7 days in urine samples with an alkaline pH. Adjustment of alkaline urine samples to a pH of 3-5 with HCl or 4.8-5.4 with citric acid completely blocked degradation of catecholamines. Ethylenediaminetetraacetic acid/metabisulphite, although reducing the extent of degradation of catecholamines in alkaline urine, was largely ineffectual as a stabiliser. Citric acid is equally effective as HCl for stabilisation of urinary free catecholamines and minimises hazards associated with use of strong inorganic acids while avoiding deconjugation of sulphate-conjugated metabolites during simultaneous LC-MS/MS measurements of free catecholamines and their free O-methylated metabolites.

  2. Simultaneous quantitative profiling of 20 isoprostanoids from omega-3 and omega-6 polyunsaturated fatty acids by LC-MS/MS in various biological samples. (United States)

    Dupuy, Aude; Le Faouder, Pauline; Vigor, Claire; Oger, Camille; Galano, Jean-Marie; Dray, Cédric; Lee, Jetty Chung-Yung; Valet, Philippe; Gladine, Cécile; Durand, Thierry; Bertrand-Michel, Justine


    Isoprostanoids are a group of non-enzymatic oxygenated metabolites of polyunsaturated fatty acids. It belongs to oxylipins group, which are important lipid mediators in biological processes, such as tissue repair, blood clotting, blood vessel permeability, inflammation and immunity regulation. Recently, isoprostanoids from eicosapentaenoic, docosahexaenoic, adrenic and α-linolenic namely F3-isoprostanes, F4-neuroprostanes, F2-dihomo-isoprostanes and F1-phytoprostanes, respectively have attracted attention because of their putative contribution to health. Since isoprostanoids are derived from different substrate of PUFAs and can have similar or opposing biological consequences, a total isoprostanoids profile is essential to understand the overall effect in the testing model. However, the concentration of most isoprostanoids range from picogram to nanogram, therefore a sensitive method to quantify 20 isoprostanoids simultaneously was formulated and measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The lipid portion from various biological samples was extracted prior to LC-MS/MS evaluation. For all the isoprostanoids LOD and LOQ, and the method was validated on plasma samples for matrix effect, yield of extraction and reproducibility were determined. The methodology was further tested for the isoprostanoids profiles in brain and liver of LDLR(-/-) mice with and without docosahexaenoic acid (DHA) supplementation. Our analysis showed similar levels of total F2-isoprostanes and F4-neuroprostanes in the liver and brain of non-supplemented LDLR(-/-) mice. The distribution of different F2-isoprostane isomers varied between tissues but not for F4-neuroprostanes which were predominated by the 4(RS)-4-F4t-neuroprostane isomer. DHA supplementation to LDLR(-/-) mice concomitantly increased total F4-neuroprostanes levels compared to F2-isoprostanes but this effect was more pronounced in the liver than brain. Copyright © 2016 Elsevier B.V. All rights

  3. Comparative Proteomic Profile of the Human Umbilical Cord Blood Exosomes between Normal and Preeclampsia Pregnancies with High-Resolution Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Ruizhe Jia


    Full Text Available Background/Aims: Exosomes are extracellular vesicles that are involved in several biological processes. The roles of proteins from human umbilical cord blood exosomes in the pathogenesis of preeclampsia remains poorly understood. Methods: In this study, we used high-resolution LC-MS/MS technologies to construct a comparative proteomic profiling of human umbilical cord blood exosomes between normal and preeclamptic pregnancies. Results: A total of 221 proteins were detected in human umbilical cord blood exosomes, with 14 upregulated and 15 downregulated proteins were definitively identified between preeclamptic and control pregnancies. Further bioinformatics analysis (Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that these differentially expressed proteins correlate with enzyme regulator activity, binding, extracellular region, cell part, biological regulation, cellular process and complement and coagulation cascades occurring during pathological changes of preeclampsia. Conclusion: Our results show significantly altered expression profiles of proteins in human umbilical cord blood exosomes between normal and preeclampsia pregnancies. These proteins may be involved in the etiology of preeclampsia.

  4. LC-MS/MS method for the determination of clodronate in human plasma. (United States)

    Hasan, Mahmoud; Schumacher, Gitta; Seekamp, Anne; Taedken, Tobias; Siegmund, Werner; Oswald, Stefan


    Clodronate belongs to the class of bisphosphonates which are used for the treatment of bone disorders. Due to its high polarity it has a low and highly variable oral bioavailability which results in low plasma concentrations and requires sensitive bioanalytical methods to characterize its pharmacokinetics in human. Here, we describe for the first time the development and validation of a LC-MS/MS assay for the quantification of clodronate in human plasma. The bisphosphonate was isolated from the biological matrix by protein precipitation using perchloric acid (10%), and derivatized with trimethylorthoacetate prior sample clean-up with liquid-liquid extraction using methyl tert-butyl ether. The chromatography was performed using an isocratic elution with ammonium acetate 5mM (85% v/v, pH 3.8) and acetonitrile (15% v/v) as mobile phase with a flow rate of 300μl/min on a reversed-phase column (Supelco Ascentis(®), C18) temporized at 50°C. The mass spectrometric detection was done using the API4000 triple quadruple mass spectrometer monitoring the mass/charge transitions 301.0/145 for clodronate and 305.2/137.1 for the internal standard etidronate. The analytical range was set to 5-800ng/ml, allowing an evaluation of the plasma concentration-time profiles of clodronate for approximately 7-8 half-life (∼24h). The method was validated according to current FDA/EMA guidelines on bioanalytical method validation with respect to specificity, linearity, intra- and inter-day accuracy and precision, matrix effect, recovery as well as stability. The precision of the assay was 0.6-6.9% and 0.6-8.1% for the intra-day and inter-day variability, respectively. The intra-day and inter-day accuracy (error) was 0.6-8.8% and 2.2-4.5%. The recovery of the analyte was low (2-3%) but reproducible over the entire validation range and sufficient to monitor the target concentrations in human plasma. The drug was shown to be stable in plasma at room temperature for at least 3h (96.0±6%) and

  5. Entwicklung einer LC/MS/MS- Methode zur Analytik polarer Pflanzenschutzmittel-Wirkstoffe und ihrer Metabolite in Erntegütern


    Pantiru, Monica Elena


    In dieser Arbeit wurde eine LC/MS/MS-Analysenmethode zur gemeinsamen Bestimmung von über 100 polaren Pflanzenschutzmitteln in Erntegütern und Lebensmitteln entwickelt. Für die sehr unterschiedlichen Substanzen aus den Gruppen der Carbamate, Harnstoffderivate, sauren Wirkstoffe, Organophosphor-Verbindungen und Triazole wurde ein einheitliches Aufarbeitungsverfahren erarbeitet. Nach Extraktion mit Methanol und Reinigung auf einer Festphase wurden die Substanzen mittels LC/MS/MS mit zwei verschi...

  6. Study on the polymorphism of G-quadruplexes by reversed-phase HPLC and LC-MS. (United States)

    Qiao, Jun-Qin; Cao, Zhao-Ming; Liang, Chao; Chen, Hong-Juan; Zheng, Wei-Juan; Lian, Hong-Zhen


    Polymorphism is inherent for G-quadruplexes (G4s), and the different structural forms are important for the participation in different biological functions of telomeres. A lot of progress has been made in the exploration of G4 polymorphism. However, quick separation and reliable assessment methods for different conformations of G4 are still very few. In this work, the polymorphism of three sequences d[(G 3 T) 3 G 3 ], d[(G 3 C) 3 G 3 ] and d[(G 3 T) 4 ] annealed in six different solutions were investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS), fluorescence spectroscopy, circular dichroism spectroscopy, together with native-polyacrylamide gel electrophoresis. Different G4 conformations of these three sequences can be separated clearly by RP-HPLC, and further characterized by on-line LC-MS analysis. It is revealed that high-order structures other than intramolecular quadruplexes were favored for d[(G 3 T) 3 G 3 ] and d[(G 3 C) 3 G 3 ] under the annealing conditions. However, flanking loop impeded d[(G 3 T) 4 ] to form higher-order structures than dimer. In addition, the nature and concentration of cation, as well as the annealing solution component, all have decent influence on the stability and relative ratios of various G4 building blocks. Based on the above findings, RP-HPLC and LC-MS combined with spectroscopic techniques can be used as a facile and powerful tool for quick separation and identification of G4s in solutions, and for effective assessment of DNA sequences and annealing environments on G4 polymorphism. The established protocol provides a novel strategy for evaluating G4 polymorphism, which will facilitate studies on quadruplex structures and their biophysical properties. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. A LC-MS/MS method for the specific, sensitive, and simultaneous quantification of 5-aminolevulinic acid and porphobilinogen. (United States)

    Zhang, Jinglan; Yasuda, Makiko; Desnick, Robert J; Balwani, Manisha; Bishop, David; Yu, Chunli


    Accurate determinations of 5-aminolevulinic acid (ALA) and porphobilinogen (PBG) in physiologic fluids are required for the diagnosis and therapeutic monitoring of acute porphyrias. Current colorimetric methods are insensitive and over-estimate ALA and PBG due to poor specificity, while LC-MS/MS methods increase sensitivity, but have limited matrices. An LC-MS/MS method was developed to simultaneously determine ALA and PBG concentrations in fluids or tissues which were solid phase extracted, butanol derivatized, and quantitated by selective reaction monitoring using (13)C(5), (15)N-ALA and 2,4-(13)C(2)-PBG internal standards. ALA was separated from interfering compounds on a reverse phase C8-column. For ALA and PBG, the matrix effects (87.3-105%) and process efficiencies (77.6-97.8% and 37.2-41.6%, respectively) were acceptable in plasma and urine matrices. The assay was highly sensitive for ALA and PBG (LLOQ=0.05 μM with 25 μL urine or 100 μL plasma), and required ∼4 h from extraction to results. ALA and PBG accuracy ranged from 88.2 to 110% (n=10); intra- and inter-assay coefficients of variations were PBG levels when asymptomatic, and high levels during acute attacks, which decreased with hemin therapy. In AIP mice, baseline ALA and PBG levels in urine, plasma, and liver were increased after phenobarbital induction 28-/63-, 42-/266-, and 13-/316-fold, respectively. This LC-MS/MS method is rapid, specific, highly sensitive, accurate, and simultaneously measures ALA and PBG in urine, plasma, and tissues permitting porphyria clinical diagnoses, therapeutic monitoring, and research. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. New 3D-printed sorbent for extraction of steroids from human plasma preceding LC-MS analysis. (United States)

    Konieczna, Lucyna; Belka, Mariusz; Okońska, Magdalena; Pyszka, Magdalena; Bączek, Tomasz


    In recent years, there has been an increasing worldwide interest in the use of alternative sample preparation methods that are proceeded by separation techniques. Fused deposition modeling (FDM) is a 3D printing technique that is based the consecutive layering of softened/melted thermoplastic materials. In this study, a group of natural steroids and sexual hormones - namely, aldosterone, cortisol, β-estradiol, testosterone, dihydrotestosterone, and synthetic methyltestosterone and betamethasone - were separated and determined using an optimized high-performance liquid chromatography coupled to mass spectrometry (LC-MS) method in positive ionization mode. 3D-printed sorbents were selected as the pre-concentration technique because they are generally low cost, fast, and simple to make and automate. Furthermore, the use of 3D-printed sorbents helps to minimize potential errors due to their repeatability and reproducibility, and their ability to eliminate carry over by using one printed sorbent for a single extraction of steroids from biological matrices. The extraction procedure was optimized and the parameters influencing 3D-printed Layfomm 60 ® based sorbent and LC-MS were studied, including the type of extraction solvent used, sorption and desorption times, temperature, and the salting-out effect. To demonstrate this method's applicability for biological sample analysis, the SPME-LC-MS method was validated for its ability to simultaneously quantify endogenous steroids. This evaluation confirmed good linearity and an R 2 that was between 0.9970 and 0.9990. The recovery rates for human plasma samples were 86.34-93.6% for the studied steroids with intra- and inter-day RSDs of 1.44-7.42% and 1.44-9.46%, respectively. To our knowledge, this study is the first time that 3D-printed sorbents have been used to extract trace amounts of endogenous low-molecular-weight compounds, such as steroids, from biological samples, such as plasma. Copyright © 2018 Elsevier B.V. All

  9. Reported prevalence and quantitative LC-MS methods for the analysis of veterinary drug residues in honey: a review. (United States)

    Venable, Ryan; Haynes, Carion; Cook, Jo Marie


    Insect pollination increases the value and productivity of three-quarters of crop species grown for food. Declining beehive health in commercial apiaries has resulted in numerous reports from government laboratories worldwide of contamination with antimicrobial chemicals in honey. This review includes pertinent discussion of legislation and events leading to increased government oversight in the commercial honey market. A detailed summary of the variety and prevalence of veterinary drug residues being found in honey as well as a selection of robust quantitative and confirmatory LC-MS methods with an emphasis on those adopted by government testing laboratories are presented.

  10. Analysis of the constituents in jojoba wax used as a food additive by LC/MS/MS. (United States)

    Tada, Atsuko; Jin, Zhe-Long; Sugimoto, Naoki; Sato, Kyoko; Yamazaki, Takeshi; Tanamoto, Kenichi


    Jojoba wax is a natural gum base used as a food additive in Japan, and is obtained from jojoba oil with a characteristically high melting point. Although the constituents of jojoba oil have been reported, the quality of jojoba wax used as a food additive has not yet been clarified. In order to evaluate its quality as a food additive and to obtain basic information useful for setting official standards, we investigated the constituents and their concentrations in jojoba wax. LC/MS analysis of the jojoba wax showed six peaks with [M+H]+ ions in the range from m/z 533.6 to 673.7 at intervals of m/z 28. After isolation of the components of the four main peaks by preparative LC/MS, the fatty acid and long chain alcohol moieties of the wax esters were analyzed by methanolysis and hydrolysis, followed by GC/MS. The results indicated that the main constituents in jojoba wax were various kinds of wax esters, namely eicosenyl octadecenoate (C20:1-C18:1) (1), eicosenyl eicosenoate (C20:1-C20:1) (II), docosenyl eicosenoate (C22:1-C20:1) (III), eicosenyl docosenoate (C20:1-C22:1) (IV) and tetracosenyl eiosenoate (C24:1-C20:1) (V). To confirm and quantify the wax esters in jojoba wax directly, LC/MS/MS analysis was performed. The product ions corresponding to the fatty acid moieties of the wax esters were observed, and by using the product ions derived from the protonated molecular ions of wax esters the fatty acid moieties were identified by MRM analysis. The concentrations of the wax esters I, II and III, in jojoba wax were 5.5, 21.4 and 37.8%, respectively. In summary, we clarified the main constituents of jojoba wax and quantified the molecular species of the wax esters without hydrolysis by monitoring their product ions, using a LC/MS/MS system.

  11. Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars

    Directory of Open Access Journals (Sweden)

    Petroczi Andrea


    Full Text Available Abstract Background Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3 and 25-hydroxyvitamin-D2 (25OHD2 collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars. Methods A positive ion electrospray ionisation (ESI LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM mode for quantification. It involved i liquid-liquid extraction, ii tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter, iii Stanozolol-D3 as internal standard, and iv identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified. Results The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3, and 7-α-hydroxy-4-cholesten-3-one (7αC4. The new assay was capable of detecting 0.25 ng/mL of all analytes in serum. Conclusions To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D. The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic

  12. Large-scale proteome comparative analysis of developing rhizomes of the ancient vascular plant Equisetum hyemale.

    Directory of Open Access Journals (Sweden)

    Tiago Santana Balbuena


    Full Text Available Equisetum hyemale is a widespread vascular plant species, whose reproduction is mainly dependent on the growth and development of the rhizomes. Due to its key evolutionary position, the identification of factors that could be involved in the existence of the rhizomatous trait may contribute to a better understanding of the role of this underground organ for the successful propagation of this and other plant species. In the present work, we characterized the proteome of E. hyemale rhizomes using a GeLC-MS spectral-counting proteomics strategy. A total of 1,911 and 1,860 non-redundant proteins were identified in the rhizomes apical tip and elongation zone, respectively. Rhizome- characteristic proteins were determined by comparisons of the developing rhizome tissues to developing roots. A total of 87 proteins were found to be up-regulated in both E. hyemale rhizome tissues in relation to developing roots. Hierarchical clustering indicated a vast dynamic range in the expression of the 87 characteristic proteins and revealed, based on the expression profile, the existence of 9 major protein groups. Gene ontology analyses suggested an over-representation of the terms involved in macromolecular and protein biosynthetic processes, gene expression and nucleotide and protein binding functions. Spatial differences analysis between the rhizome apical tip and the elongation zone revealed that only eight proteins were up-regulated in the apical tip including RNA-binding proteins and an acyl carrier protein, as well as a KH-domain protein and a T-complex subunit; while only seven proteins were up-regulated in the elongation zone including phosphomannomutase, galactomannan galactosyltransferase, endoglucanase 10 and 25 and mannose-1-phosphate guanyltransferase subunits alpha and beta. This is the first large scale characterization of the proteome of a plant rhizome. Implications of the findings were discussed in relation to other underground organs and related

  13. Mass spectrometry based proteomics, background, status and future needs

    DEFF Research Database (Denmark)

    Roepstorff, Peter


    LC-MS is described. A number of challenging problems which have been solved using different proteomics strategies including the advantage of organell enrichment or modifications specific peptide isolation to get deeper into the proteome are described. Finally the present status and future needs discussed....


    DEFF Research Database (Denmark)

    Slagel, Joseph; Deutsch, Eric; Mendoza, Luis

    in a timely manner. Compounding the problem is TPP’s iProphet which can significantly improve the confidence of identifications by combining the output of multiple search engines but then requires orders more searches to be performed. We show that cloud computing products like Amazon Web Services (AWS......) provides a viable solution to meet this need. Methods Amazon Web Services provides an especially flexible platform for enabling cloud computing applications by providing virtualized servers that are capable of executing custom virtual machine images, have almost unlimited and secure file storage...... on Amazon Web Services using the TPP command line tool amztpp. The first was a Canine dataset consisting of 982 runs from an LTQ Orbitrap organized in 35 groups. The second set is a Bovine dataset consisting of 1210 iTRAQ and non-iTRAQ runs from both a QSTAR XL and QSTAR Elite. Both datasets are processed...

  15. Simultaneous Proteomic Discovery and Targeted Monitoring using Liquid Chromatography, Ion Mobility Spectrometry, and Mass Spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Burnum-Johnson, Kristin E.; Nie, Song; Casey, Cameron P.; Monroe, Matthew E.; Orton, Daniel J.; Ibrahim, Yehia M.; Gritsenko, Marina A.; Clauss, Therese R. W.; Shukla, Anil K.; Moore, Ronald J.; Purvine, Samuel O.; Shi, Tujin; Qian, Weijun; Liu, Tao; Baker, Erin S.; Smith, Richard D.


    Current proteomics approaches are comprised of both broad discovery measurements as well as more quantitative targeted measurements. These two different measurement types are used to initially identify potentially important proteins (e.g., candidate biomarkers) and then enable improved quantification for a limited number of selected proteins. However, both approaches suffer from limitations, particularly the lower sensitivity, accuracy, and quantitation precision for discovery approaches compared to targeted approaches, and the limited proteome coverage provided by targeted approaches. Herein, we describe a new proteomics approach that allows both discovery and targeted monitoring (DTM) in a single analysis using liquid chromatography, ion mobility spectrometry and mass spectrometry (LC-IMS-MS). In DTM, heavy labeled peptides for target ions are spiked into tryptic digests and both the labeled and unlabeled peptides are broadly detected using LC-IMS-MS instrumentation, allowing the benefits of discovery and targeted approaches. To understand the possible improvement of the DTM approach, it was compared to LC-MS broad measurements using an accurate mass and time tag database and selected reaction monitoring (SRM) targeted measurements. The DTM results yielded greater peptide/protein coverage and a significant improvement in the detection of lower abundance species compared to LC-MS discovery measurements. DTM was also observed to have similar detection limits as SRM for the targeted measurements indicating its potential for combining the discovery and targeted approaches.

  16. Microcolumns with self-assembled particle frits for proteomics

    DEFF Research Database (Denmark)

    Ishihama, Yasushi; Rappsilber, Juri; Andersen, Jens S


    bridged the outlet with 0.3 pl dead volume and perfect success rate. In peptide analysis by LC-MS, the peak width at half height was normally less than 6 s, compared to 12 s without SAPs. The LC-MS-MS system provided 37% sequence coverage (21 matched peptides) for a tryptically-digested sample of 10 fmol...

  17. Symbiont diversity in Reticulitermes santonensis (Isoptera: Rhinotermitidae): investigation strategy through proteomics. (United States)

    Bauwens, Julien; Millet, Catherine; Tarayre, Cedric; Brasseur, Catherine; Destain, Jacqueline; Vandenbol, Micheline; Thonart, Philippe; Portetelle, Daniel; De Pauw, Edwin; Haubruge, Eric; Francis, Frederic


    The complex microbial community living in the hindgut of lower termites includes prokaryotes, flagellates, yeasts, and filamentous fungi. Many microorganisms are found in the termite gut, but only a few are thought to be involved in symbiotic association to participate in cellulose digestion. Proteomics provides analyses from both taxonomical and functional perspectives. We aimed to identify symbiont diversity in the gut of Reticulitermes santonensis (Feytaud), via complementary electrospray ionization associated to ion trap tandem mass spectrometry (LC-MS/MS) and two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry analysis. One specific challenge to the study of lower termites is the relatively few data available on abundant symbiotic flagellates. Analysis based on LC-MS/MS revealed few protein families showing assignments to eukaryotes and the taxonomic origin of highly represented actins could not be established. Tubulins proved to be the most suitable protein family with which to identify flagellate populations from hindgut samples using LC-MS/MS, compared with other protein families, although this method targeted few prokaryotes in our assay. Similarly, two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry did not succeed in identifying flagellate populations, but did permit the identification of most of the prokaryotic components of the symbiotic system. Finally, fungi and yeasts were identified by both methods. Owing to the lack of sequenced genes in flagellates, targeting tubulins for LC-MS/MS could allow fingerprints of flagellate populations to be established. Experimental and technical improvements might increase the efficiency of identification of prokaryotic populations in the near future, based on metaproteomic development.

  18. Liquid chromatography/mass spectrometry (LC/MS) identification of photooxidative degradates of crystalline and amorphous MK-912. (United States)

    Qin, X; Frech, P


    How and why the chemical stability of amorphous solid is different from crystalline solid is an important problem. In this study, this problem is addressed by evaluation of the photodegradation of both crystalline and amorphous MK-912 (an alpha-2 adrenoceptor antagonist) according to the photostability tests of the ICH (International Conference on Harmonization) guidelines. Under the ICH conditions, the photodegradation rate of the amorphous MK-912 was approximately 40 times faster than that of the crystalline MK-912. The photodegradation yielded isomeric, oxidative degradates. Three keto-degradates (molecular weight of 14 Da over MK-912) were observed for both forms. But, whereas five alcohol and one N-oxide degradates (molecular weight of 16 Da over MK-912) were observed for the amorphous form, only one alcohol degradate was observed for the crystalline form. Liquid chromatography/mass spectrometry (LC/MS) and LC/MS/MS were applied to identify these low-level photodegradates. A thorough analysis of the MS/MS data of protonated MK-912 was the key to the identification, and the special MS/MS features of the degradates due to the structural modifications from degradations were also important. Following this strategy, the structures of all the photodegradates were proposed. The structural identification of the photodegradates of MK-912 shed light on the different photostabilities between the crystalline and amorphous MK-912.

  19. Discrimination of Four Marine Biofilm-Forming Bacteria by LC-MS Metabolomics and Influence of Culture Parameters. (United States)

    Favre, Laurie; Ortalo-Magné, Annick; Greff, Stéphane; Pérez, Thierry; Thomas, Olivier P; Martin, Jean-Charles; Culioli, Gérald


    Most marine bacteria can form biofilms, and they are the main components of biofilms observed on marine surfaces. Biofilms constitute a widespread life strategy, as growing in such structures offers many important biological benefits. The molecular compounds expressed in biofilms and, more generally, the metabolomes of marine bacteria remain poorly studied. In this context, a nontargeted LC-MS metabolomics approach of marine biofilm-forming bacterial strains was developed. Four marine bacteria, Persicivirga (Nonlabens) mediterranea TC4 and TC7, Pseudoalteromonas lipolytica TC8, and Shewanella sp. TC11, were used as model organisms. The main objective was to search for some strain-specific bacterial metabolites and to determine how culture parameters (culture medium, growth phase, and mode of culture) may affect the cellular metabolism of each strain and thus the global interstrain metabolic discrimination. LC-MS profiling and statistical partial least-squares discriminant analyses showed that the four strains could be differentiated at the species level whatever the medium, the growth phase, or the mode of culture (planktonic vs biofilm). A MS/MS molecular network was subsequently built and allowed the identification of putative bacterial biomarkers. TC8 was discriminated by a series of ornithine lipids, while the P. mediterranea strains produced hydroxylated ornithine and glycine lipids. Among the P. mediterranea strains, TC7 extracts were distinguished by the occurrence of diamine derivatives, such as putrescine amides.

  20. Development of a screening assay for ligands to the estrogen receptor based on magnetic microparticles and LC-MS (United States)

    Choi, Yongsoo; van Breemen, Richard B.


    A high throughput screening assay for the identification of ligands to pharmacologically significant receptors was developed based on magnetic particles containing immobilized receptors followed by liquid chromatography—mass spectrometry (LC-MS). This assay is suitable for the screening of complex mixtures such as botanical extracts. For proof-of-principle, estrogen receptor-α (ER-α) and ER-β were immobilized on magnetic particles functionalized with aldehyde or carboxylic acid groups. Alternatively, biotinylated ER was immobilized onto streptavidin-derivatized magnetic particles. The ER that was immobilized using the streptavidin-biotin chemistry showed higher activity than that immobilized on aldehyde or carboxylic acid functionalized magnetic particles. Immobilized ER was incubated with extracts of Trifolium pratense L. (red clover) or Humulus lupulus L. (hops). As a control for non-specific binding, each botanical extract was incubated with magnetic particles containing no ER. After magnetic separation of the particles containing bound ligands from the unbound components in the extract, the particles were washed, ligands were released using methanol, and then the ligands were identified using LC-MS. The estrogens genistein and daidzein were identified in the red clover extract, and the estrogen 8-prenylnaringenin was identified in the hop extract. These screening results are consistent with those obtained using previous screening approaches. PMID:18220538

  1. Identification of Eupatilin from Artemisia argyi as a Selective PPARα Agonist Using Affinity Selection Ultrafiltration LC-MS

    Directory of Open Access Journals (Sweden)

    Yongsoo Choi


    Full Text Available Peroxisome proliferator-activated receptors (PPARs are key nuclear receptors and therapeutic targets for the treatment of metabolic diseases through the regulation of insulin resistance, diabetes, and dyslipidemia. Although a few drugs that target PPARs have been approved, more diverse and novel PPAR ligands are necessary to improve the safety and efficacy of available drugs. To expedite the search for new natural agonists of PPARs, we developed a screening assay based on ultrafiltration liquid chromatography-mass spectrometry (LC-MS that is compatible with complex samples such as dietary foods or botanical extracts. The known PPARα and/or PPARγ ligands resveratrol and rosiglitazone were used as positive controls to validate the developed method. When applied to the screening of an Artemisia argyi extract, eupatilin was identified as a selective PPARα ligand. A PPAR competitive binding assay based on FRET detection also confirmed eupatilin as a selective PPARα agonist exhibiting a binding affinity of 1.18 μM (IC50. Furthermore, eupatilin activation of the transcriptional activity of PPARα was confirmed using a cell-based transactivation assay. Thus, ultrafiltration LC-MS is a suitable assay for the identification of PPAR ligands in complex matrixes such as extracts of dietary foods and botanicals.

  2. Development of LC-MS determination method and back-propagation ANN pharmacokinetic model of corynoxeine in rat. (United States)

    Ma, Jianshe; Cai, Jinzhang; Lin, Guanyang; Chen, Huilin; Wang, Xianqin; Wang, Xianchuan; Hu, Lufeng


    Corynoxeine(CX), isolated from the extract of Uncaria rhynchophylla, is a useful and prospective compound in the prevention and treatment for vascular diseases. A simple and selective liquid chromatography mass spectrometry (LC-MS) method was developed to determine the concentration of CX in rat plasma. The chromatographic separation was achieved on a Zorbax SB-C18 (2.1 mm × 150 mm, 5 μm) column with acetonitrile-0.1% formic acid in water as mobile phase. Selective ion monitoring (SIM) mode was used for quantification using target ions m/z 383 for CX and m/z 237 for the carbamazepine (IS). After the LC-MS method was validated, it was applied to a back-propagation artificial neural network (BP-ANN) pharmacokinetic model study of CX in rats. The results showed that after intravenous administration of CX, it was mainly distributed in blood and eliminated quickly, t1/2 was less than 1h. The predicted concentrations generated by BP-ANN model had a high correlation coefficient (R>0.99) with experimental values. The developed BP-ANN pharmacokinetic model can be used to predict the concentration of CX in rats. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Screening for anabolic steroids in urine of forensic cases using fully automated solid phase extraction and LC-MS-MS. (United States)

    Andersen, David W; Linnet, Kristian


    A screening method for 18 frequently measured exogenous anabolic steroids and the testosterone/epitestosterone (T/E) ratio in forensic cases has been developed and validated. The method involves a fully automated sample preparation including enzyme treatment, addition of internal standards and solid phase extraction followed by analysis by liquid chromatography-tandem mass spectrometry (LC-MS-MS) using electrospray ionization with adduct formation for two compounds. Urine samples from 580 forensic cases were analyzed to determine the T/E ratio and occurrence of exogenous anabolic steroids. Extraction recoveries ranged from 77 to 95%, matrix effects from 48 to 78%, overall process efficiencies from 40 to 54% and the lower limit of identification ranged from 2 to 40 ng/mL. In the 580 urine samples analyzed from routine forensic cases, 17 (2.9%) were found positive for one or more anabolic steroids. Only seven different steroids including testosterone were found in the material, suggesting that only a small number of common steroids are likely to occur in a forensic context. The steroids were often in high concentrations (>100 ng/mL), and a combination of steroids and/or other drugs of abuse were seen in the majority of cases. The method presented serves as a fast and automated screening procedure, proving the suitability of LC-MS-MS for analyzing anabolic steroids. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  4. High sensitivity LC-MS/MS method for direct quantification of human parathyroid 1-34 (teriparatide) in human plasma. (United States)

    Chambers, Erin E; Lame, Mary E; Bardsley, Jon; Hannam, Sally; Legido-Quigley, Cristina; Smith, Norman; Fountain, Kenneth J; Collins, Eileen; Thomas, Elizabeth


    Teriparatide, the 1-34 fragment of human parathyroid hormone, is used to treat osteoporosis patients with a high risk of fracture by stimulating new bone formation. Routinely teriparatide is quantified using radioimmunoassay however the LC-MS/MS described here has the potential to achieve greater accuracy and precision, higher specificity, and is readily implemented in routine bioanalytical laboratories. Hence a complete method combining effective sample prep with appropriate LC separation and selected reaction monitoring (SRM) MS detection was developed to selectively separate teriparatide from closely related endogenous peptides and to reduce interferences. Samples were concentrated without evaporation, minimizing the risk of adsorptive losses. Chromatography was performed on a sub 2μm particle charged surface hybrid column, which provided significantly higher peak capacity than a traditional C18 column when formic acid was used as the mobile phase modifier. Total LC cycle time was 6min. An LOD of 15pg/mL (3.6fmol/mL) from 200μL of human plasma was readily achieved and standard curves were accurate and precise from 15pg/mL to 500pg/mL. Mean QC accuracies ranged from 90% to 106%. Mean QC precision was better than 7%. The CV of matrix factors across 6 sources of human plasma was 5%. The assay presented here is the first LC-MS method which reaches clinically relevant detection limits for teriparatide. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Studying the effect of storage conditions on the metabolite content of red wine using HILIC LC-MS based metabolomics. (United States)

    Arapitsas, Panagiotis; Corte, Anna Della; Gika, Helen; Narduzzi, Luca; Mattivi, Fulvio; Theodoridis, Georgios


    The main aim of this work was to develop an untargeted normal phase LC-MS method, starting from a targeted method already validated for the analysis of 135 polar metabolites. Since the LC instrument and column were the same, most of the chromatographic conditions remained identical, while the adaptations focused on maintaining the ionic strength of the eluents constant. The sample preparation was simplified and the effectiveness of LC-MS for long batches was evaluated, in order to record the maximum number of metabolites with good chromatographic resolution and the best MS stability and accuracy. The method was applied to study the influence of storage conditions on wine composition. Slightly sub-optimum storage conditions had a major impact on the polar metabolite fingerprint of the red wines analysed and the markers revealed included phenolics, vitamins and metabolites indentified in wine for the first time (4-amino-heptanedioic acid and its ethyl ester). Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. LC-MS/MS determination of betamethasone and its phosphate and acetate esters in human plasma after sample stabilization. (United States)

    Salem, Isam I; Alkhatib, Musab; Najib, Naji


    Two specific liquid chromatography-mass spectrometric (LC-MS/MS) assays were developed and validated for the determination of betamethasone (BET), and its acetate (BA) and phosphate (BP) esters. The plasma and the blood used for the development and validation of these two methods were previously stabilized. Liquid-liquid extraction techniques were used after the addition of prednisolone as internal standard (IS). Samples were chromatographed using C8 column, while mass detection was carried out by electrospray ionization in the positive mode (ESI+). The method was proved linear over a working range 0.50-50.00 ng/ml for BET (r(2)>0.99), while BA linear range was 1.0-20.0 ng/ml (r(2)>0.99). Sensitivity was determined as 0.50 ng/ml for BET and 1.00 ng/ml for BA. Betamethasone phosphate LC-MS/MS method involved solid phase extraction after the addition of prednisolone phosphate as (IS). Separation was carried out using C18 column, while detection was by ESI+. The method showed good linearity over the working range 2.0-200.0 ng/ml (r(2)>099). Both methods were applied to determine BET, BA and BP in plasma samples obtained for pharmacokinetics studies in human. Copyright © 2011 Elsevier B.V. All rights reserved.

  7. Determination of 14 aminoglycosides by LC-MS/MS using molecularly imprinted polymer solid phase extraction for clean-up. (United States)

    Savoy, Marie-Claude; Woo, Pei Mun; Ulrich, Pauline; Tarres, Adrienne; Mottier, Pascal; Desmarchelier, Aurélien


    An LC-MS/MS method for screening 14 aminoglycosides in foodstuffs of animal origin is presented. Its scope includes raw materials and processed ingredients but also finished products composed of milk, meat, fish, egg or fat. Aminoglycosides are extracted in an acidic aqueous solution, which is first recovered after centrifugation, then diluted with a basic buffer and finally purified by molecularly imprinted polymer-solid phase extraction (MIP-SPE). Analytes are detected within 8 min by ion-pair reversed phase LC-MS/MS. Due to the large range of foodstuffs involved, the variability of matrix effects led to significant MS signal variations. This was circumvented by systematically extracting each sample twice, i.e. 'unspiked' and 'spiked' at the screening target concentration of 50 µg kg -1 . The method was validated according to the European Community Reference Laboratories Residues Guidelines giving false-negative and false-positive rates ≤3% for all compounds. Ruggedness of the method was further demonstrated in quality control operations by a second laboratory. The 14 aminoglycosides in water-based standard solutions were stable for up to 6 months when stored at either -80°C, -20°C or at 4°C storage temperatures.

  8. Measurement of ampicillin residue levels in chicken eggs during and after medicated feed administration by LC-MS/MS. (United States)

    Hamamoto, Kouko; Akama, Ryoko; Mizuno, Yasuharu


    A simple and sensitive LC-MS/MS method was developed and validated for the determination of ampicillin (ABPC) in chicken eggs. Residues were extracted by reverse-phase solid-phase extraction. Chromatographic separation was performed using a reverse-phase column with an elution gradient. The limits of detection and quantification were 0.01 and 0.1 ng g(-1), respectively. For the 0.1-50 ng g(-1) concentration range, mean recovery and accuracy values were 93.9-98.5% and 100.2-118.0%, respectively. ABPC residue concentrations in eggs before, during and after 7 days of medicated feeding of maximum dosage (40 mg kg(-1) body weight day(-1)) of ABPC were determined with the LC-MS/MS method. The maximum concentration of ABPC in eggs was 3.6 ± 1.7 ng g(-1) (mean ± SD) on the last day of the administration period. Residue concentrations of ABPC in eggs during and after ABPC administration were not over the Japanese maximum residue limit of 0.01 mg kg(-1).

  9. Detection of endocrine active substances in the aquatic environment in southern Taiwan using bioassays and LC-MS/MS. (United States)

    Chen, Kuang-Yu; Chou, Pei-Hsin


    Endocrine active substances, including naturally occurring hormones and various synthetic chemicals have received much concern owing to their endocrine disrupting potencies. It is essential to monitor their environmental occurrence since these compounds may pose potential threats to biota and human health. In this study, yeast-based reporter assays were carried out to investigate the presence of (anti-)androgenic, (anti-)estrogenic, and (anti-)thyroid compounds in the aquatic environment in southern Taiwan. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was also used to measure the environmental concentrations of selected endocrine active substances for assessing potential ecological risks and characterizing contributions to the endocrine disrupting activities. Bioassay results showed that anti-androgenic (ND-7489 μg L(-1) flutamide equivalent), estrogenic (ND-347 ng L(-1) 17β-estradiol equivalent), and anti-thyroid activities were detected in the dissolved and particulate phases of river water samples, while anti-estrogenic activities (ND-10 μg L(-1) 4-hydroxytamoxifen equivalent) were less often found. LC-MS/MS analysis revealed that anti-androgenic and estrogenic contaminants, such as bisphenol A, triclosan, and estrone were frequently detected in Taiwanese rivers. In addition, their risk quotient values were often higher than 1, suggesting that they may pose an ecological risk to the aquatic biota. Further identification of unknown anti-androgenic and estrogenic contaminants in Taiwanese rivers may be necessary to protect Taiwan's aquatic environment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Analysis of acidic endogenous phytohormones in grapes by using online solid-phase extraction coupled with LC-MS/MS. (United States)

    Yu, Jian-Na; Meng, Qing-Yan; Liu, Wen-Jie; Lu, Ya-Ling; Ren, Xiao-Lin


    Phytohormones play important roles in regulating numerous plant physiological and developmental processes, even during the postharvest storage period. In order to determine the functions and changes of gibberellins acid (GA3), indoleacetic acid (IAA), abscisic acid (ABA), indolebutyric acid (IBA) and jasmonic acid (JA) in grape berries during storage, an ultrasensitive method based on direct injection online solid-phase extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) was developed. Grape berries were extracted with cold methanol. After centrifugation, the supernatants were concentrated with a vacuum centrifugal concentrator and injected into an online solid-phase extraction column. After the cleanup procedure, the analytes were determined by LC-MS/MS. The results showed that the linearity of the proposed method was 10-210 µg kg(-1) for ABA, 20-200 µg kg(-1) for IBA, 15-320 µg kg(-1) for IAA, 20-320 µg kg(-1) for GA3 and 3.0-90.0 µg kg(-1) for JA. The limits of detection of the method were 0.71, 2.79, 0.94, 0.39 and 0.57 µg kg(-1), respectively. The proposed method was successfully applied to the analysis of endogenous phytohormones in grape berries during the postharvest storage period. © The Author [2013]. Published by Oxford University Press. All rights reserved. For Permissions, please email:

  11. HILIC LC-MS for the determination of 2'-C-methyl-cytidine-triphosphate in rat liver. (United States)

    Pucci, Vincenzo; Giuliano, Claudio; Zhang, Rena; Koeplinger, Kenneth Alan; Leone, Joseph F; Monteagudo, Edith; Bonelli, Fabio


    A very accurate and selective LC-MS/MS method was developed and validated for the quantification of 2'-C-modified nucleoside triphosphate in liver tissue samples. An efficient pretreatment procedure of liver tissue samples was developed, using a fully automated SPE procedure with 96-well SPE plate (weak anion exchange sorbent, 30 mg). Nucleotide hydrophilic interaction chromatography has been performed on an aminopropyl column (100 mm x 2.0 mm, 3 microm) using a gradient mixture of ACN and ACN/water (5:95 v/v) with 20 mM ammonium acetate at pH 9.45 as mobile phase at 300 microL/min flow rate. The 2'-C-modified nucleoside triphosphate was detected in the negative ESI mode in multiple reaction monitoring (MRM) mode. Calibration curve was linear over the 0.05-50 microM concentration range. Satisfying results, confirming the high reliability of the established LC-MS/MS method, were obtained for intraday precision (CV = 2.5-9.1%) and accuracy (92.6-94.8%) and interday precision (CV = 9.6-11.5%) and accuracy (94.4-102.4%) as well as for recovery (82.0-112.6%) and selectivity. The method has been successfully applied for pharmacokinetic studies of 2'-C-methyl-cytidine-triphosphate in liver tissue samples.

  12. A LC-MS-MS method for determination of low doxazosin concentrations in plasma after oral administration to dogs. (United States)

    Erceg, Marijana; Cindric, Mario; Pozaic Frketic, Lidija; Vertzoni, Maria; Cetina-Cizmek, Biserka; Reppas, Christos


    A rapid and sensitive reversed phase liquid chromatography- tandem mass spectrometry (LC-MS-MS) method is developed for the determination of doxazosin in canine plasma. The samples are prepared by precipitation of proteins using a mixture of methanol and acetonitrile, followed by freezing and evaporation of the organic solvent. The remaining dry residue is redissolved in mobile phase and analyzed by LC-MS-MS with positive electrospray ionization using the selected reactions monitoring mode. An XTerra MS C(18) column, a mobile phase composed of acetonitrile and 2mM ammonium acetate with gradient elution, and a flow rate of 400 microL/min are employed. The elution times for prazosin (internal standard) and doxazosin are approximately 8 and 10 min, respectively. Calibration curves are linear in the 1-20 ng/mL concentration range. Limits of detection and quantification are 0.4 ng/mL and 1.2 ng/mL, respectively. Recovery is higher than 94%. Intra- and inter-day relative standard deviations are below 7% and 8%, respectively. The method is applied for the determination of doxazosin plasma levels following a single administration of doxazosin base and doxazosin mesylate tablets (2 mg dose) to dogs in the fed state. The results indicate possible superiority of the mesylate salt on the plasma input rates of doxazosin.

  13. Determination of Acid Herbicides Using Modified QuEChERS with Fast Switching ESI(+)/ESI(-) LC-MS/MS. (United States)

    Sack, Chris; Vonderbrink, John; Smoker, Michael; Smith, Robert E


    A method for the determination of 35 acid herbicides in food matrices was developed, validated, and implemented. It utilizes a modified QuEChERS extraction procedure coupled with quantitation by liquid chromatography tandem mass spectrometry (LC-MS/MS). The acid herbicides analyzed are all organic carboxylic acids, including the older chlorophenoxy acid herbicides such as 2,4-dichlorophenoxyacetic acid (2,4-D), dicamba, 4-chlorophenoxyacetic acid (4-CPA), quinclorac, and many of the newer imidazolinone herbicides such as imazethapyr and imazaquin. In the procedure, 10 mL of water is added to 5 g of sample and then extracted with 1% formic acid in acetonitrile for 1 min. The acetonitrile phase is salted out of the extract by adding sodium chloride and magnesium sulfate, followed by centrifugation. The acetonitrile is diluted 1:1 with water to enable quantitation by LC-MS/MS using fast switching between positive and negative electrospray ionization modes. The average recoveries for all the compounds except aminocyclopyrachlor were 95% with a precision of 8%. The method detection limits for all residues were less than 10 ng/g, and the correlation coefficients for the calibration curves was greater than 0.99 for all but two compounds tested. The method was used successfully for the quantitation of acid herbicides in the FDA's total diet study. The procedure proved to be accurate, precise, linear, sensitive, and rugged.

  14. Punica granatum peel extracts: HPLC fractionation and LC MS analysis to quest compounds having activity against multidrug resistant bacteria. (United States)

    Khan, Ilyas; Rahman, Hazir; Abd El-Salam, Nasser M; Tawab, Abdul; Hussain, Anwar; Khan, Taj Ali; Khan, Usman Ali; Qasim, Muhammad; Adnan, Muhammad; Azizullah, Azizullah; Murad, Waheed; Jalal, Abdullah; Muhammad, Noor; Ullah, Riaz


    Medicinal plants are rich source of traditional herbal medicine around the globe. Most of the plant's therapeutic properties are due to the presence of secondary bioactive compounds. The present study analyzed the High Pressure Liquid Chromatography (HPLC) fractions of Puncia granatum (peel) extracts (aqueous, chloroform, ethanol and hexane) against multidrug resistant bacterial pathogens (Acinetobacter baumannii, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus). All the fractions having antibacterial activity was processed for bioactive compounds identification using LC MS/MS analysis. Among total HPLC fractions (n = 30), 4 HPLC fractions of P. granatum (peel) showed potential activity against MDR pathogens. Fraction 1 (F1) and fraction 4 (F4) collected from aqueous extract showed maximum activity against P. aeruginosa. Fraction 2 (F2) of hexane showed antibacterial activity against three pathogens, while ethanol F4 exhibited antibacterial activity against A. baumannii. The active fractions were processed for LC MS/MS analysis to identify bioactive compounds. Valoneic acid dilactone (aqueous F1 and F4), Hexoside (ethanol F4) and Coumaric acid (hexane F2) were identified as bioactive compounds in HPLC fractions. Puncia granatum peel extracts HPLC fractions exhibited potential inhibitory activity against MDR bacterial human pathogens. Several bioactive compounds were identified from the HPLC fractions. Further characterization of these compounds may be helpful to conclude it as therapeutic lead molecules against MDR pathogens.

  15. SIMPATIQCO: a server-based software suite which facilitates monitoring the time course of LC-MS performance metrics on Orbitrap instruments. (United States)

    Pichler, Peter; Mazanek, Michael; Dusberger, Frederico; Weilnböck, Lisa; Huber, Christian G; Stingl, Christoph; Luider, Theo M; Straube, Werner L; Köcher, Thomas; Mechtler, Karl


    While the performance of liquid chromatography (LC) and mass spectrometry (MS) instrumentation continues to increase, applications such as analyses of complete or near-complete proteomes and quantitative studies require constant and optimal system performance. For this reason, research laboratories and core facilities alike are recommended to implement quality control (QC) measures as part of their routine workflows. Many laboratories perform sporadic quality control checks. However, successive and systematic longitudinal monitoring of system performance would be facilitated by dedicated automatic or semiautomatic software solutions that aid an effortless analysis and display of QC metrics over time. We present the software package SIMPATIQCO (SIMPle AuTomatIc Quality COntrol) designed for evaluation of data from LTQ Orbitrap, Q-Exactive, LTQ FT, and LTQ instruments. A centralized SIMPATIQCO server can process QC data from multiple instruments. The software calculates QC metrics supervising every step of data acquisition from LC and electrospray to MS. For each QC metric the software learns the range indicating adequate system performance from the uploaded data using robust statistics. Results are stored in a database and can be displayed in a comfortable manner from any computer in the laboratory via a web browser. QC data can be monitored for individual LC runs as well as plotted over time. SIMPATIQCO thus assists the longitudinal monitoring of important QC metrics such as peptide elution times, peak widths, intensities, total ion current (TIC) as well as sensitivity, and overall LC-MS system performance; in this way the software also helps identify potential problems. The SIMPATIQCO software package is available free of charge.

  16. A Novel Algorithm for Validating Peptide Identification from a Shotgun Proteomics Search Engine (United States)

    Jian, Ling; Niu, Xinnan; Xia, Zhonghang; Samir, Parimal; Sumanasekera, Chiranthani; Zheng, Mu; Jennings, Jennifer L.; Hoek, Kristen L.; Allos, Tara; Howard., Leigh M.; Edwards, Kathryn M.; Weil, P. Anthony; Link, Andrew J.


    Liquid chromatography coupled with tandem mass spectrometry has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC/MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm based on the resolution and mass accuracy of the mass spectrometer employed in the LC/MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines. PMID:23402659

  17. Identification of N-glycosylation in hepatocellular carcinoma patients' serum with a comparative proteomic approach.

    Directory of Open Access Journals (Sweden)

    Yingnan Huang

    Full Text Available AIM: This study is to explore the different expressions of serum N-glycoproteins and glycosylation sites between hepatocellular carcinoma (HCC patients and healthy controls. METHOD: We combined high abundant proteins depletion and hydrophilic affinity method to enrich the glycoproteins. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS, we extensively surveyed different expressions of glycosylation sites and glycoproteins between the two groups. RESULT: This approach identified 152 glycosylation sites and 54 glycoproteins expressed differently between HCC patients and healthy controls. With the absolute values of Pearson coefficients of at least 0.8, eight proteins were identified significantly up or down regulated in HCC serum. Those proteins are supposed to be involved in several biological processes, cellular components and molecular functions of hepatocarcinogenesis. Several of them had been reported abnormally regulated in several kinds of malignant tumors, and may be promising biomarkers of HCC. CONCLUSION: Our work provides a systematic and quantitative method of glycoproteomics and demonstrates some key changes in clinical HCC serum. These proteomic signatures may help to unveil the underlying mechanisms of hepatocarcinogenesis and may be useful for the exploration of candidate biomarkers.

  18. Development and optimization of simplified LC-MS/MS quantification of 25-hydroxyvitamin D using protein precipitation combined with on-line solid phase extraction (SPE). (United States)

    Thibeault, Denis; Caron, Nicolas; Djiana, Rose; Kremer, Richard; Blank, David


    25-Hydroxyvitamin D, the most useful marker of the vitamin D status of an individual, has seen an exponential growth of its routine measurement in recent years. Several methods are currently offered but the most specific is LC-MS/MS. However, the routine use of this technique in the clinical laboratory makes it essential to improve key steps of this method for high throughput delivery. Importantly, the preanalytical steps of this assay and the efficacy of the separation system need to be optimized prior to MS detection. In this report we replaced the standard and time consuming liquid-liquid extraction method of vitamin D metabolites with hexane (LLE) combined with centrifugation (LLE/centrifugation) by a simpler protein precipitation with extraction (PPE) in acetonitrile combined with a fast separation process using a 96-well plate filtration system (PPE/filtration). This rapid extraction was then followed by an on-line solid phase extraction (SPE) using a selective chromatographic separation. We also optimized the operational and consumable costs, by using an inexpensive guard column as a trapping column to significantly enhance the lifespan of the analytical column two to three times as compared to conventional chromatography. The LC-MS/MS technique permits the measurement of both 25-hydroxyvitamin D(2) (25-OH D(2)) and the 25-hydroxyvitamin D(3) (25-OH D(3)) metabolites in electrospray ionization (ESI) mode. The chromatographic system consisted of a 2.1 mm × 50 mm C18 3.5 μM column with a 2.1 mm × 20 mm C18 3.5 μM guard column connected with two 6 ports switching valves. Quantifications were done using the isotopic dilution technique with hexadeutered 25-OH D(3) and 25-OH D(2).The ion suppression problem with phospholipids was also evaluated and optimized to minimize this effect through the chromatography process and the on-line SPE trapping. Calibration curves were prepared by diluting a commercial high calibrator Chromsystems (München, Germany) with

  19. Evaluation of cell factory performance through determination of intracellular metabolites using LC-MS/MS

    DEFF Research Database (Denmark)

    Magdenoska, Olivera; Martinussen, Jan; Nielsen, Kristian Fog


    combination of sensitivity and specificity of this technique allowed qualitative and quantitative analysis of low metabolite concentrations. The mains steps involved were: (i) metabolite sample preparation by quenching, extraction, sample purification using dispersive Solid Phase Extraction; (ii) quantitative......A major objective in biotechnology is the improvement of the efficiency of host microorganisms used as cell factories. Engineering a strain capable of producing high amounts of a desired biochemical is a multi-step process consisting of design, construction, and analysis of the constructed cell...... factory. In order to address the function or disfunction of the engineered cells,systems biology tools are employed by using the multi “omics” approach (genomics, transcriptomics, proteomics, metabolomics and fluxomics). Metabolomics is a tool aimed at a quantitative understanding of metabolism...

  20. An adaptive alignment algorithm for quality-controlled label-free LC-MS. (United States)

    Sandin, Marianne; Ali, Ashfaq; Hansson, Karin; Månsson, Olle; Andreasson, Erik; Resjö, Svante; Levander, Fredrik


    Label-free quantification using precursor-based intensities is a versatile workflow for large-scale proteomics studies. The method however requires extensive computational analysis and is therefore in need of robust quality control during the data mining stage. We present a new label-free data analysis workflow integrated into a multiuser software platform. A novel adaptive alignment algorithm has been developed to minimize the possible systematic bias introduced into the analysis. Parameters are estimated on the fly from the data at hand, producing a user-friendly analysis suite. Quality metrics are output in every step of the analysis as well as actively incorporated into the parameter estimation. We furthermore show the improvement of this system by comprehensive comparison to classical label-free analysis methodology as well as current state-of-the-art software.

  1. Comparative transcriptomic and proteomic profiling of industrial wine yeast strains. (United States)

    Rossouw, Debra; van den Dool, Adri H; Jacobson, Dan; Bauer, Florian F


    The geno- and phenotypic diversity of commercial Saccharomyces cerevisiae wine yeast strains provides an opportunity to apply the system-wide approaches that are reasonably well established for laboratory strains to generate insight into the functioning of complex cellular networks in industrial environments. We have previously analyzed the transcriptomes of five industrial wine yeast strains at three time points during alcoholic fermentation. Here, we extend the comparative approach to include an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomic analysis of two of the previously analyzed wine yeast strains at the same three time points during fermentation in synthetic wine must. The data show that differences in the transcriptomes of the two strains at a given time point rather accurately reflect differences in the corresponding proteomes independently of the gene ontology (GO) category, providing strong support for the biological relevance of comparative transcriptomic data sets in yeast. In line with previous observations, the alignment proves to be less accurate when assessing intrastrain changes at different time points. In this case, differences between the transcriptome and proteome appear to be strongly dependent on the GO category of the corresponding genes. The data in particular suggest that metabolic enzymes and the corresponding genes appear to be strongly correlated over time and between strains, suggesting a strong transcriptional control of such enzymes. The data also allow the generation of hypotheses regarding the molecular origin of significant differences in phenotypic traits between the two strains.

  2. Core-shell LC-MS/MS method for quantification of second generation anticoagulant rodenticides diastereoisomers in rat liver in relationship with exposure of wild rats. (United States)

    Fourel, Isabelle; Damin-Pernik, Marlène; Benoit, Etienne; Lattard, Virginie


    Second generation anticoagulant rodenticides (SGARs), pesticides used worldwide to control rodent populations, exist in two diastereoisomer chemical species because they own two stereogenic centers. A core-shell LC-MS/MS multi-residue method for comprehensive quantitative analysis of the diastereoisomers of five SGARs as well as three first generation anticoagulant rodenticide molecules has been fully validated in liver of rats according to a bioanalytical guideline. A core-shell column (superficially porous particles) has been chosen for its ability to separate the diastereomers of bromadiolone, difenacoum, brodifacoum, flocoumafen and difethialone and for its robustness to rat liver extracts. The highly selective chromatographic separation of the diastereoisomers contributes to good signal to noise ratios and then enhances the sensitivity of the method compared to the ones of fully porous columns. An elution gradient has been optimized with 10mM ammonium acetate and acetonitrile as aqueous/organic mobile phase respectively. Triple quadrupole mass detector has been used to achieve specifity and LLOQ from 0.92 to 2.2ng/g for each diastereoisomer, or first generation anticoagulant rodenticides. Then we evidenced diastereoisomeric ratios in liver of rats issued from not controlled exposure of wild rats (Rattus norvegicus) trapped in a French Parisian park through a campaign of rodent eradication. We compared them to diastereoisomeric ratios in SGARs commercial baits that contain both isomers, and showed that one of the two diastereoiomers had nearly disappeared in liver of rats. The proportions of cis-bromadiolone and trans-difenacoum were really lowered compared to the baits: 5/7 and 9/12 rats had only trans-bromadiolone and cis-difenacoum hepatic residues respectively. Liver persistence of the two diastereoisomers of bromadiolone and difenacoum was different due to differences in their pharmacokinetics in wild rats. The new core-shell LC-MS/MS method is

  3. Development of Chiral LC-MS Methods for small Molecules and Their Applications in the Analysis of Enantiomeric Composition and Pharmacokinetic Studies

    Energy Technology Data Exchange (ETDEWEB)

    Desai, Meera Jay [Iowa State Univ., Ames, IA (United States)


    The purpose of this research was to develop sensitive LC-MS methods for enantiomeric separation and detection, and then apply these methods for determination of enantiomeric composition and for the study of pharmacokinetic and pharmacodynamic properties of a chiral nutraceutical. Our first study, evaluated the use of reverse phase and polar organic mode for chiral LC-API/MS method development. Reverse phase methods containing high water were found to decrease ionization efficiency in electrospray, while polar organic methods offered good compatibility and low limits of detection with ESI. The use of lower flow rates dramatically increased the sensitivity by an order of magnitude. Additionally, for rapid chiral screening, the coupled Chirobiotic column afforded great applicability for LC-MS method development. Our second study, continued with chiral LC-MS method development in this case for the normal phase mode. Ethoxynonafluorobutane, a fluorocarbon with low flammability and no flashpoint, was used as a substitute solvent for hexane/heptane mobile phases for LC-APCI/MS. Comparable chromatographic resolutions and selectivities were found using ENFB substituted mobile phase systems, although, peak efficiencies were significantly diminished. Limits of detection were either comparable or better for ENFB-MS over heptane-PDA detection. The miscibility of ENFB with a variety of commonly used organic modifiers provided for flexibility in method development. For APCI, lower flow rates did not increase sensitivity as significantly as was previously found for ESI-MS detection. The chiral analysis of native amino acids was evaluated using both APCI and ESI sources. For free amino acids and small peptides, APCI was found to have better sensitivities over ESI at high flow rates. For larger peptides, however, sensitivity was greatly improved with the use of electrospray. Additionally, sensitivity was enhanced with the use of non-volatile additives, This optimized method was then

  4. Comparative proteomic analysis in Miscanthus sinensis exposed to antimony stress

    International Nuclear Information System (INIS)

    Xue, Liang; Ren, Huadong; Li, Sheng; Gao, Ming; Shi, Shengqing; Chang, Ermei; Wei, Yuan; Yao, Xiaohua; Jiang, Zeping; Liu, Jianfeng


    To explore the molecular basis of Sb tolerance mechanism in plant, a comparative proteomic analysis of both roots and leaves in Miscanthus sinensis has been conducted in combination with physiological and biochemical analyses. M. sinensis seedlings were exposed to different doses of Sb, and both roots and leaves were collected after 3 days of treatment. Two-dimensional gel electrophoresis (2-DE) and image analyses found that 29 protein spots showed 1.5-fold change in abundance in leaves and 19 spots in roots, of which 31 were identified by MALDI-TOF-MS and MALDI-TOF-TOF-MS. Proteins involved in antioxidant defense and stress response generally increased their expression all over the Sb treatments. In addition, proteins relative to transcription, signal transduction, energy metabolism and cell division and cell structure showed a variable expression pattern over Sb concentrations. Overall these findings provide new insights into the probable survival mechanisms by which M. sinensis could be adapting to Sb phytotoxicity. - Highlights: • Proteomics in Miscanthus sinensis leaves and roots exposed to Sb stress were studied. • There were 31 spots that were identified by mass spectrometry. • Most of these proteins were involved in antioxidant defense and stress response. • Our findings provide new insights into the tolerant mechanisms to Sb stress. - Miscanthus sinensis proteomic analysis under Sb stress reveals probable molecular mechanisms on Sb detoxification

  5. Enhancement of sensitivity in the determination of organic trace compounds in complex matrices with liquid chromatography-mass spectrometry (LC-MS)

    International Nuclear Information System (INIS)

    Mascher, D.G.


    The PhD-thesis deals with 'enhancement of sensitivity in the determination of organic trace compounds in complex matrices with liquid chromatography-mass spectrometry (LC-MS)'. Almost the most important factor is the enhancement of the ionization yield with Atmospheric Pressure Chemical Ionization (APCI) or Electrospray Ionization (ESI) in LC-MS. Ionization yields of different compounds can vary by a factor of 10000. Three ways to solve this problem of little ionization yield were tried: 1) Modification of the mobile phase in HPLC 2) Chemical modification of the analytes 3) A new type of ionization called Atmospheric Pressure Photo Ionization (APPI). ad 1) By using specific additives to mobile phases ion suppression that might derive from an ion pair reagent that was necessary for chromatography could be omitted. General remarks cannot be done. ad 2) Chemical modification or so called derivatization is well known for UV- and fluorescence-detection for a long period of time. As substances containing nitrogene (e.g. primary, secondary or tertiary amines) often have good ionization yields, poor or relatively poor ionizable substances like carboxylic acids, sugars and partially phenolic steroids were used as analytes for derivatization reactions. By using Dansyl as Dansylchlorid or Dansylhydrazine a basic derivatization agent could be found that ionizes very well. A 200 times more sensitive determination of estrogenes is possible after derivatization with Dansylchlorid. Using a tandem-mass-spectrometer a lower limit of quantification of 2 pg/mL plasma could be reached by using 1 mL of plasma. For ketones like carvon and campher an enhancement by a factor of 500 and 4000 could be reached by using Dansylhydrazine as the derivatization agent. For fatty acids DMEQ as derivatization agent enhanced the sensitivity by a factor of 20 to 100. ad 3) APPI as a new ionization mode showed really good results for specific molecules. Relatively unpolar substances as diphenylsulfide

  6. Comparison of two-concentration with multi-concentration linear regressions: Retrospective data analysis of multiple regulated LC-MS bioanalytical projects. (United States)

    Musuku, Adrien; Tan, Aimin; Awaiye, Kayode; Trabelsi, Fethi


    Linear calibration is usually performed using eight to ten calibration concentration levels in regulated LC-MS bioanalysis because a minimum of six are specified in regulatory guidelines. However, we have previously reported that two-concentration linear calibration is as reliable as or even better than using multiple concentrations. The purpose of this research is to compare two-concentration with multiple-concentration linear calibration through retrospective data analysis of multiple bioanalytical projects that were conducted in an independent regulated bioanalytical laboratory. A total of 12 bioanalytical projects were randomly selected: two validations and two studies for each of the three most commonly used types of sample extraction methods (protein precipitation, liquid-liquid extraction, solid-phase extraction). When the existing data were retrospectively linearly regressed using only the lowest and the highest concentration levels, no extra batch failure/QC rejection was observed and the differences in accuracy and precision between the original multi-concentration regression and the new two-concentration linear regression are negligible. Specifically, the differences in overall mean apparent bias (square root of mean individual bias squares) are within the ranges of -0.3% to 0.7% and 0.1-0.7% for the validations and studies, respectively. The differences in mean QC concentrations are within the ranges of -0.6% to 1.8% and -0.8% to 2.5% for the validations and studies, respectively. The differences in %CV are within the ranges of -0.7% to 0.9% and -0.3% to 0.6% for the validations and studies, respectively. The average differences in study sample concentrations are within the range of -0.8% to 2.3%. With two-concentration linear regression, an average of 13% of time and cost could have been saved for each batch together with 53% of saving in the lead-in for each project (the preparation of working standard solutions, spiking, and aliquoting). Furthermore

  7. Matrix removal in state of the art sample preparation methods for serum by charged aerosol detection and metabolomics-based LC-MS. (United States)

    Schimek, Denise; Francesconi, Kevin A; Mautner, Anton; Libiseller, Gunnar; Raml, Reingard; Magnes, Christoph


    Investigations into sample preparation procedures usually focus on analyte recovery with no information provided about the fate of other components of the sample (matrix). For many analyses, however, and particularly those using liquid chromatography-mass spectrometry (LC-MS), quantitative measurements are greatly influenced by sample matrix. Using the example of the drug amitriptyline and three of its metabolites in serum, we performed a comprehensive investigation of nine commonly used sample clean-up procedures in terms of their suitability for preparing serum samples. We were monitoring the undesired matrix compounds using a combination of charged aerosol detection (CAD), LC-CAD, and a metabolomics-based LC-MS/MS approach. In this way, we compared analyte recovery of protein precipitation-, liquid-liquid-, solid-phase- and hybrid solid-phase extraction methods. Although all methods provided acceptable recoveries, the highest recovery was obtained by protein precipitation with acetonitrile/formic acid (amitriptyline 113%, nortriptyline 92%, 10-hydroxyamitriptyline 89%, and amitriptyline N-oxide 96%). The quantification of matrix removal by LC-CAD showed that the solid phase extraction method (SPE) provided the lowest remaining matrix load (48-123 μg mL(-1)), which is a 10-40 fold better matrix clean-up than the precipitation- or hybrid solid phase extraction methods. The metabolomics profiles of eleven compound classes, comprising 70 matrix compounds showed the trends of compound class removal for each sample preparation strategy. The collective data set of analyte recovery, matrix removal and matrix compound profile was used to assess the effectiveness of each sample preparation method. The best performance in matrix clean-up and practical handling of small sample volumes was showed by the SPE techniques, particularly HLB SPE. CAD proved to be an effective tool for revealing the considerable differences between the sample preparation methods. This detector can

  8. Determination of a beta(3)-agonist in human plasma by LC/MS/MS with semi-automated 48-well diatomaceous earth plate. (United States)

    Wang, A Q; Fisher, A L; Hsieh, J; Cairns, A M; Rogers, J D; Musson, D G


    Methods for the determination of a beta(3)-agonist (A) in human plasma were developed and compared based on high-performance liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection using a turbo ion spray (TIS) interface. Drug and internal standard were isolated from plasma by three sample preparation methods, liquid-liquid extraction, Chem Elut cartridges and 48-well diatomaceous earth plates, that successively improved sample throughput for LC/MS/MS. MS/MS detection was performed on a PE Sciex API 365 tandem mass spectrometer operated in positive ion mode and using multiple reaction monitoring (MRM). The precursor/product ion combinations of m/z 625/607 and 653/515 were used to quantify A and internal standard, respectively, after chromatographic separation of the analytes. Using liquid-liquid extraction and Chem Elut cartridges, the assay concentration range was 0.5-100 ng/ml. Using diatomaceous earth plates, the concentration range of the assay was extended to 0.5-200 ng/ml. For all three assays, the statistics for precision and accuracy is comparable. The assay accuracy ranged from 91-107% and intraday precision as measured by the coefficient of variation (CV) ranged 2-10%. The sample throughput was tripled when the diatomaceous earth plate method was compared with the original liquid-liquid extraction method.

  9. The Probabilistic Convolution Tree: Efficient Exact Bayesian Inference for Faster LC-MS/MS Protein Inference (United States)

    Serang, Oliver


    Exact Bayesian inference can sometimes be performed efficiently for special cases where a function has commutative and associative symmetry of its inputs (called “causal independence”). For this reason, it is desirable to exploit such symmetry on big data sets. Here we present a method to exploit a general form of this symmetry on probabilistic adder nodes by transforming those probabilistic adder nodes into a probabilistic convolution tree with which dynamic programming computes exact probabilities. A substantial speedup is demonstrated using an illustration example that can arise when identifying splice forms with bottom-up mass spectrometry-based proteomics. On this example, even state-of-the-art exact inference algorithms require a runtime more than exponential in the number of splice forms considered. By using the probabilistic convolution tree, we reduce the runtime to and the space to where is the number of variables joined by an additive or cardinal operator. This approach, which can also be used with junction tree inference, is applicable to graphs with arbitrary dependency on counting variables or cardinalities and can be used on diverse problems and fields like forward error correcting codes, elemental decomposition, and spectral demixing. The approach also trivially generalizes to multiple dimensions. PMID:24626234

  10. Antioxidant Activity of Flaxseed (Linum usitatissimum L. shell and Analysis of Its Polyphenol Contents by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Hatica Han


    Full Text Available Flaxseed (Linum usitatissimum L. is important source of oil and protein for industrial, pharmaceutical, and nutritional applications. In order to estimate the effects of lyophilized aqueous extract of flaxseed shell (AEF and evaporated ethanolic extract of flaxseed shell (EEF, we studied their DPPH, ABTS, DMPD and O 2 •- scavenging effects. Total antioxidant activity by ferric thiocyanate method, Fe 3+, Cu 2+ and [Fe 3+-(TPTZ 2] 3+ reducing ability, and Fe 2+ chelating activity. Also, α-tocopherol, BHA, trolox, and BHT were used as positive controls. The results clearly AEF and EEF demonstrated effective antioxidant activity. The quantity of p-hydroxybenzoic, vanillin, p-coumaric acid, ascorbic acid, ferulic acid, and ellagic acid were investigated by LC-MS/MS. The present study will introduce a novelty for further studies on the antioxidant effects of AEF and EEF.

  11. Simultaneous determination of metronidazole and spiramycin I in human plasma, saliva and gingival crevicular fluid by LC-MS/MS. (United States)

    Sagan, Cyriaque; Salvador, Arnaud; Dubreuil, Didier; Poulet, Pierre P; Duffaut, D; Brumpt, Ivan


    An analytical validation of a new liquid chromatographic-mass spectrometric (LC-MS/MS) method for simultaneous determination of metronidazole and spiramycin I concentrations in human plasma, saliva and gingival crevicular fluid (GCF) is presented. Ornidazole was used as an internal standard, and sample pre-treatment consisted of a liquid-liquid extraction. Chromatographic separation was achieved on a 5 microm Kromasil C18 column (150 mm x 4.6 mm i.d., particle size 5 microm), with a gradient using acetonitrile, water and formic acid at a flow rate of 0.9 ml/min. The methods were validated in terms of intra- and inter-batch precision (methods are applicable for accurate and simultaneous monitoring of the plasma, saliva and gingival crevicular fluid levels of metronidazole and spiramycin I from pharmacokinetic studies.

  12. Selection of possible signature peptides for the detection of bovine lactoferrin in infant formulas by LC-MS/MS.

    Directory of Open Access Journals (Sweden)

    Mingmei Yuan

    Full Text Available An LC-MS/MS assay based on a signature peptide was developed and fully validated for the quantitation of bovine lactoferrin in infant formulas. Three unreported signature peptides were derived and identified from the tryptic peptides of bovine lactoferrin. The peptide ETTVFENLPEK was used for quantification based on assay performance. The blank matrix camel milk powder and bovine lactoferrin protein standards were mixed and spiked with stable isotope-labeled internal standard to establish a calibration curve. The established method was extensively validated by determining the linearity (R2 > 0.999, sensitivity (limit of quantitation, 0.16 mg/100 g, recovery (83.1-91.6%, precision (RSD < 5.4% and repeatability (RSD < 7.7%. To validate the applicability of the method, four different brands of infant formulas in China were analysed. The acquired contents of bovine lactoferrin were 52.60-150.56 mg/100 g.

  13. Skin sample preparation by collagenase digestion for diclofenac quantification using LC-MS/MS after topical application. (United States)

    Nirogi, Ramakrishna; Padala, Naga Surya Prakash; Boggavarapu, Rajesh Kumar; Kalaikadhiban, Ilayaraja; Ajjala, Devender Reddy; Bhyrapuneni, Gopinadh; Muddana, Nageswara Rao


    Skin is the target site to evaluate the pharmacokinetic parameters of topical applications. Sample preparation is one of the influential steps in the bioanalysis of drugs in the skin. Evaluation of dermatopharmacokinetics at preclinical stage is challenging due to lack of proper sample preparation method. There is a need for an efficient sample preparation procedure for quantification of drugs in the skin using LC-MS/MS. The skin samples treated with collagenase followed by homogenization using a bead beater represents a best-fit method resulting in uniform homogenate for reproducible results. A new approach involving enzymatic treatment and mechanical homogenization techniques were evaluated for efficient sample preparation of skin samples in the bioanalysis.

  14. Identification of Datura Species Involved in a Food-Poisoning Case Using LC-MS/MS and DNA Barcording. (United States)

    Ushiyama, Atsuko; Akaboshi, Chie; Ohsawa, Nobuhiko; Shimizu, Tomomi; Matsushima, Yuki; Shimizu, Hideaki; Hashiguchi, Shigeki


    A food-poisoning case due to eating the roots of Datura occurred in Kawasaki City, Japan in 2014. The Datura plant was mistakenly collected instead of burdock in a domestic garden. The roots of these plants are quite similar to each other. We presumed that the specimen was the root of Datura, but it was difficult to classify it only from the morphology. Using LC-MS/MS, we detected atropine and scopolamine from the remaining plant specimen. Therefore, we applied the DNA barcoding method. The results showed that the specimen was classified into Solanaceae family, but not Asteraceae family. Thus, the specimen was confirmed to be Datura species based on both chemical and genetic analyses.

  15. A rapid LC-MS/MS quantification of peramivir using a simple and inexpensive sample precipitation: application to PK. (United States)

    Wang, Zhan-Zhang; Zhang, Ming; Shang, De-Wei; Ni, Xiao-Jia; Hu, Jin-Qing; Qiu, Chang; Wen, Yu-Guan


    Peramivir is a newly approved selective neuraminidase inhibitor designed to inhibit influenza virus infection. We report a robust and sensitive method utilizing simple precipitation extraction with LC-MS/MS for the high-throughput quantification. Addition of 0.06 M of ammonium formate and 0.1% formic acid in mobile phase could help reduce the matrix effect. This method uses 100 µl of plasma and covers a linear concentration range from 5 to 10,000 ng/ml. Other validation parameters are also evaluated and meet regulatory expectations by US FDA guidelines. The developed HPLC-MS/MS method has been successfully applied to support a clinical pharmacokinetic study. The strategy presented here can be applied elsewhere and may be useful for other amphiphilic drugs.

  16. Determination of dimethyltryptamine and β-carbolines (ayahuasca alkaloids) in plasma samples by LC-MS/MS. (United States)

    Oliveira, Carolina Dizioli Rodrigues; Okai, Guilherme Gonçalves; da Costa, José Luiz; de Almeida, Rafael Menck; Oliveira-Silva, Diogo; Yonamine, Mauricio


    Ayahuasca is a psychoactive plant beverage originally used by indigenous people throughout the Amazon Basin, long before its modern use by syncretic religious groups established in Brazil, the USA and European countries. The objective of this study was to develop a method for quantification of dimethyltryptamine and β-carbolines in human plasma samples. The analytes were extracted by means of C18 cartridges and injected into LC-MS/MS, operated in positive ion mode and multiple reaction monitoring. The LOQs obtained for all analytes were below 0.5 ng/ml. By using the weighted least squares linear regression, the accuracy of the analytical method was improved at the lower end of the calibration curve (from 0.5 to 100 ng/ml; r(2)> 0.98). The method proved to be simple, rapid and useful to estimate administered doses for further pharmacological and toxicological investigations of ayahuasca exposure.

  17. Determination of 14 monoalkyl phosphates, dialkyl phosphates and dialkyl thiophosphates by LC-MS/MS in human urinary samples. (United States)

    Reemtsma, Thorsten; Lingott, Jana; Roegler, Stefanie


    Human urine was analyzed for nine dialkyl (DAP) and five monoalkyl phosphates (MAP) by LC-MS/MS. Some phosphoric acid esters are industrial chemicals and other hydrolysis products of trialkyl or triaryl phosphates, used as pesticides, flame retardants or plasticizers. Five MAP and two DAP were detected here for the first time in human urine. Monobutyl, diethyl, diphenyl and diethylhexyl phosphate were determined with median concentrations in the μg/L-range. The total urinary concentration of the 14 DAP and MAP summed up to a median of 20μg/L. Inclusion of MAP in future biomonitoring studies should provide a more comprehensive picture of the exposure of humans to organophosphorus compounds. Copyright © 2011 Elsevier B.V. All rights reserved.

  18. Simple and rapid determination of unsaturated fatty acids in 1 µl of rat plasma by LC-MS/MS. (United States)

    Zheng, Shirui; Ma, Zhiyuan; Song, Feifeng; Ye, Jianfeng; Wang, Ruwei; Tu, Meijuan; Li, Liping; Zhou, Hui; Zeng, Su; Jiang, Huidi


    Deficiency or imbalance of unsaturated fatty acids will promote the pathogenesis of many diseases. In order to monitor the exposure of unsaturated fatty acids, the method based on LC-MS/MS was developed. Standard calibration curves for α-linolenic acid, linoleic acid, palmitoleic acid and oleic acid were linear (r ≥0.99). The intra-and interbatch accuracy (RE%) ranged from -4.5 to 8.6%, while the intra- and interbatch precisions (RSD%) were ≤8.7%. The extraction recovery varied from 85.4 to 99.6%, and no obvious matrix effect was observed. The method offers a simple approach for measuring 4 unsaturated fatty acids in 1 μl rat plasma within 3.95 min.

  19. A novel ion pairing LC/MS metabolomics protocol for study of a variety of biologically relevant polar metabolites. (United States)

    Knee, Jose M; Rzezniczak, Teresa Z; Barsch, Aiko; Guo, Kevin Z; Merritt, Thomas J S


    We report a method of ion-pairing liquid chromatography coupled to mass spectrometry (IP-LC-MS) that we have developed for the sensitive detection and quantification of a variety of biologically relevant polar molecules. We use the ion-pairing agent diamyl ammonium to improve chromatographic resolution of polar compounds, such as nucleotide cofactors, sugar phosphates, and organic acids, that are generally poorly retained by conventional reverse phase chromatographic methods. This method showed good linearity (average R value of 0.996) and reproducibility (generally RSD values <10%). We demonstrate the utility of this method by investigating the metabolomic signature of three distinct biological systems: the metabolic response to lack of superoxide dismutase activity and to paraquat induced oxidative stress, and the metabolic profiles of four different Drosophila species. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Lysozyme oxidation by singlet molecular oxygen: Peptide characterization using [18 O]-labeling oxygen and nLC-MS/MS. (United States)

    Marques, Emerson Finco; Medeiros, Marisa H G; Di Mascio, Paolo


    Singlet molecular oxygen ( 1 O 2 ) is generated in biological systems and reacts with different biomolecules. Proteins are a major target for 1 O 2 , and His, Tyr, Met, Cys, and Trp are oxidized at physiological pH. In the present study, the modification of lysozyme protein by 1 O 2 was investigated using mass spectrometry approaches. The experimental findings showed methionine, histidine, and tryptophan oxidation. The experiments were achieved using [ 18 O]-labeled 1 O 2 released from thermolabile endoperoxides in association with nano-scale liquid chromatography coupled to electrospray ionization mass spectrometry. The structural characterization by nLC-MS/MS of the amino acids in the tryptic peptides of the proteins showed addition of [ 18 O]-labeling atoms in different amino acids. Copyright © 2017 John Wiley & Sons, Ltd.

  1. Determination of zeranol, taleranol, zearalanone, α-zearalenol, β-zearalenol and zearalenone in urine by LC-MS/MS. (United States)

    Matraszek-Zuchowska, Iwona; Wozniak, Barbara; Zmudzki, Jan


    An LC-MS/MS method was developed for the sensitive and selective determination of zeranol, taleranol, zearalanone, α-zearalenol, β-zearalenol and zearalenone in animal urine. Analysis was performed on the free compounds after enzymatic deconjugation. Sample preparation included liquid-liquid extraction followed by solid-phase extraction (SPE) with C18 and NH2 columns. For chromatographic separation of hormones an Inertsil(®) ODS-3 analytical column (150 mm × 2.1 mm, 3 µm) was used. The analytes were determined and identified by LC-MS/MS on a QTRAP5500 instrument with a TurboIon-Spray source operating in negative electrospray ionisation mode. For confirmatory purposes at least two transitions were obtained for each analyte. According to Commission Decision 2002 /657/EC the validation parameters - recovery, repeatability, reproducibility, linearity, specificity, decision limits and detection capabilities - were determined. All parameters were in agreement with 2002/657/EC performance criteria. The apparent recovery ranged from 76.2% to 116.3% for all examined compounds. The repeatability was below 20% and reproducibility did not exceed the limit of 25% for most analytes. Linearity was good for all analytes in the whole range of tested concentrations, as proved by the correlation coefficients greater than 0.99. The decision limits (CCα) ranged from 0.04 to 0.18 μg l(-1) for all analytes whereas the detection capabilities (CCβ) ranged from 0.07 to 0.31 μg l(-1), respectively. All CCα and CCβ values were below the recommended concentration of 2 μg l(-1). This analytical method will be used in an integrated system for Polish monitoring programmes for the confirmation of violative screened samples.

  2. LC-MS Guided Isolation of Bioactive Principles from Iris hookeriana and Bioevaluation of Isolates for Antimicrobial and Antioxidant Activities. (United States)

    Dar, B A; Lone, S H; Shah, W A; Bhat, K A


    The genus Iris is diverse both in the abundance of secondary metabolities as well as the biological activities. The rhizomes of Iris hookeriana exhibit significant anthelminthic activity against gastrointestinal nematodes of sheep. Although Iris hookeriana has been a subject of the study of so many phytochemical studies, yet we report some constituents for the first time from this plant using a different isolation approach. This manuscript presents the isolation, antimicrobial and antioxidant evaluation of bioactive principles from Iris hookeriana. LC-MS guided isolation technique was applied for the separation of target constituents. The isolates were characterised by spectral techniques and subjected to antioxidant evaluation by DPPH assay. Four compounds; resveratrol, resveratroloside, junipeginin C and isorhamnetin-3-O-neohesperidoside were isolated for the first time along with 3 known compounds viz piceid, irigenin and iridin from I. hookeriana using this approach. The antioxidant activity screening of the isolates revealed that all the 4 constituents isolated for the first time, have strong antioxidant potential with IC50 of 14.0 µg/ml (resveratroloside), 19.7 µg/ml (junipeginen C), 12.8 µg/ml (resveratrol) and 19.8 µg/ml (isorhamnetin-3-O-neohesperodoside). So it can be safely concluded that LC-MS guided isolation of chemical compounds from Iris hookeriana has furnished 4 antioxidant constituents. Thus Iris hookeriana can act as as a good source of wonder molecule resveratrol and its 2 glycosides, resveratrolside and piceid which upon hydrolysis can be converted into the parent drug resveratrol. © Georg Thieme Verlag KG Stuttgart · New York.

  3. [Determination of brucine and strychnine in rat after cutaneous administration of semen strychni niosome gel by LC-MS/MS]. (United States)

    Li, Jingya; Miao, Fengru; Lin, Li; Zhu, Dan; Lin, Chengren; Liu, Jianxun


    A sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method has been developed and validated for the determination of brucine and strychnine in rat plasma. Samples were extracted by ethyl acetate-n-butanol (7: 3). Chromatographic separation was operated on ZORBAX XDB-C18 column with gradient elution of acetonitrile-methanol-water (0.05% acetic acid and 10 nmol x L(-1) ammonium formate contained), followed by LC-MS/MS in positive electrospray ionization. Quantification was carried out on multiple reaction monitoring (MRM) of the transition m/z 395.2/324.2, m/z 335.2/184.2 and m/z 199.1/171.1 for brucine, strychnine and tacrine (internal standard), respectively. The method was linear in the range of 0.195-100 and 0.07840 microg x L(-1) for brucine and strychnine, with coefficient correlation 0.994 and 0.996 respectively. The recoveries of extraction were 78.9% - 102.4% for brucine and 95.2% - 106.1% for strychnine. Precision, accuracy, stability and matrix effect of the analytes met the requirement. The method was applied to a pharmacokinetic study of brucine and strychnine after cutaneous administration of Semen Strychni niosome gel. The C(max) were (26.20 +/- 5.81) and (12.50 +/- 3.00) microg x L(-1) while the AUC(0-infinity), were (193.75 +/- 39.43) and (98.25 +/- 28.54) microg x h x L(-1) of the two components. We conclude that the niosomes may reduce the systemic exposures and prolong the local release of brucine and strychnine.

  4. A selective detection of lysophosphatidylcholine in dried blood spots for diagnosis of adrenoleukodystrophy by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Ryuichi Mashima


    Full Text Available X-linked adrenoleukodystrophy (X-ALD is a rare inherited metabolic disorder characterized by an impaired beta-oxidation of very long chain fatty acids in the peroxisomes. Recent studies have suggested that 1-hexacosanoyl-2-hydroxy-sn-glycero-3-phosphocholine (Lyso-PC 26:0 can be a sensitive biomarker for X-ALD. Although approximately 10-fold increase in the concentration of Lyso-PC 26:0 in DBSs from X-ALD-affected individuals were reported, whether the carriers might be distinguished from the healthy controls remained unclear. To address this question, we have validated previously developed LC-MS/MS-based analytical procedures using QC DBS. We found that the recovery of Lyso-PC 26:0 from the QC DBSs was 73.6 ± 0.3% when 2 μM of Lyso-PC 26:0 was spiked into the blood. Based on this result, the amounts of Lyso-PC 26:0 in the controls and ALD-affected individuals were 0.090 ± 0.004 (n = 11 and 1.078 ± 0.217 (n = 4 pmol/DBS, respectively. Interestingly, the concentration of Lyso-PC 26:0 in the carriers were 0.548 ± 0.095 pmol/DBS (n = 3, indicating that the carriers and the healthy controls can be distinguished. These results suggest that LC-MS/MS-based technique can be used for the detection of asymptomatic carriers and X-ALD-affected subjects in the newborn screening.

  5. Evaluation of matrix effect in determination of some bioflavonoids in food samples by LC-MS/MS method. (United States)

    Ćirić, Andrija; Prosen, Helena; Jelikić-Stankov, Milena; Đurđević, Predrag


    In the present work the LC-MS/MS method with solid phase extraction for simultaneous determination of bioflavonoids rutin, quercetin, hesperidin, hesperetin and kaempferol in some food samples (red onion, orange peel and honey) was developed and the matrix effect accompanying this determination was quantified. The matrix effect evaluated using a postextraction addition method was found to be negative in the range -44 to -0.5%, indicating ionization suppression and strongly depended on bioflavonoid concentration. The observed matrix effect was explained taking into account the co-elution of phenolic acids, in terms of their acid-base and hydrophilic properties. The efficacy of extraction expressed as the absolute recoveries of flavonoids were 88-96%, indicating very good efficiency of extraction. The extracts of food samples obtained either by Soxhlet or ultrasonic extraction were analyzed for bioflavonoid content by the LC-MS/MS method in selected reaction monitoring mode using a triple quadrupole detector and standard addition method, which was found to be the most suitable calibration approach for these samples. The optimized separation was achieved on a Phenomenex Gemini C18 column with gradient elution and mobile phase composition A: 2% acetic acid in water and B: acetonitrile. R(s) values were in the range from 1.3 to 3.1, indicating good selectivity of the method. The obtained results (mg/100g fresh weight) for different bioflavonids were for rutin 0.16, for quercetin in the range 0.65-56, for hesperidin 0.016-24, for hesperetin 0.0068-36.4 and for kaempferol 0.14-1.63 and generally show good agreement with published data. Low detection limits (0.014-0.063 μg/mL) were obtained with acceptable recoveries (86-114%). Total time of analysis was less than 40 min, therefore the proposed method represents significant improvement over existing methods. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. [Study on methodology of determination of Yizhi osmotic pump tablets active ingredient in Beagle dog plasma by LC-MS/MS]. (United States)

    Wang, Yanming; Du, Shouying; Zhai, Yongsong; Lu, Yang


    To develop a sensitive and specific LC-MS/MS method for determination of Yizhi osmotic pump tablets active ingredient in Beagle dog plasma. Beagle dog plasma pre-treatment methods were established. Geniposide, notoginsenoside R1, ginsenoside Rg1 and Rb1 notoginsenoside molecular ions and fragment ions peaks were separated well and detected synchronously by LC-MS/MS with digoxin as internal standard. Under the selected LC-MS/MS conditions, the characteristic fragment ions of the four components could be well separated and quantified, and the calibration curves showed good linearity within a certain concentration range of each component; extraction recoveries of those four compounds in plasma were higher than 75%, method recoveries were higher than 90%; day precision (RSD active ingredients in Beagle dog plasma.

  7. Identification of amino acid epimerization and isomerization in crystallin proteins by tandem LC-MS. (United States)

    Tao, Yuanqi; Julian, Ryan R


    Post-translational modifications that do not result in a change in mass are particularly difficult to detect by mass spectrometry. For example, isomerization of aspartic acid or epimerization of any chiral residue within a peptide do not lead to mass shifts but can be identified by examination of independently acquired tandem mass spectra or by combination with another technique. For analysis of a biological sample, this means that liquid chromatography or some other type of separation must be used to first separate the isomers from one another. Furthermore, each specific m/z of interest must be sampled repeatedly to allow for comparison of the tandem mass spectra from each separated isomer, which contrasts with the traditional approach in proteomics where the goal is typically to avoid resampling the same m/z. We illustrate that isomerization and epimerization of peptides can be identified in this fashion by examination of long-lived crystallin proteins extracted from a sheep eye lens. Tandem mass spectrometry relying on a combination of radical directed dissociation (RDD) and collision induced dissociation (CID) following separation by liquid chromatography was used to identify modified peptides. Numerous sites of isomerization and epimerization, including several that have not been previously identified, were determined with peptide specificity. It is demonstrated that the specific sites of amino acid isomerization within each peptide can be identified by comparison with synthetic peptides. For α-crystallin proteins, the sites that undergo the greatest degree of isomerization correspond to disordered regions, which may have important implications on chaperone functionality within the context of aging.

  8. A LC-MS method allowing the analysis of HMX and RDX present at the picogram level in natural aqueous samples without a concentration step. (United States)

    Vigneau, Olivier; Machuron-Mandard, Xavier


    The introduction of chloroform into the nebulising gas of a LC/MS electrospray interface (ESI), in a perfectly controlled way, leads to the formation of intense adducts ([M+Cl](-)) when a mobile phase containing HMX (1,3,5,7-tetranitro-1,3,5,7-tetrazacyclooctane or octogen) and RDX (1,3,5-trintro-1,3,5-triazacyclohexane or hexogen) is eluted. This LC/MS method allows the direct analysis of aqueous samples containing HMX and RDX at the pictogram level without a concentration step. The method is used to determine HMX and RDX concentrations in ground water samples from a military site.

  9. Serum concentrations of DHEA, DHEAS, 17α-hydroxyprogesterone, Δ4-androstenedione and testosterone in children determined by TurboFlow-LC-MS/MS

    DEFF Research Database (Denmark)

    Søeborg, T; Frederiksen, H; Fruekilde, Palle


    Diagnosis and management of infants and children with sex steroid disorders require fast and simultaneous assessment of several sex steroid metabolites in serum at low concentrations and on small sample volumes. Therefore, we developed a sensitive and selective TurboFlow-LC-MS/MS method for quant......Diagnosis and management of infants and children with sex steroid disorders require fast and simultaneous assessment of several sex steroid metabolites in serum at low concentrations and on small sample volumes. Therefore, we developed a sensitive and selective TurboFlow-LC-MS/MS method...

  10. The simultaneous detection and quantification of p-aminobenzoic acid and its phase 2 biotransformation metabolites in human urine using LC-MS/MS. (United States)

    Nortje, Carla; Jansen van Rensburg, Peet; Cooke, Cecile; Erasmus, Elardus


    p-Aminobenzoic acid (PABA) can be used as a probe substance to investigate glycine conjugation, a reaction of phase 2 biotransformation. An LC-MS/MS method for simultaneous quantification of PABA and its metabolites from human urine was developed and validated. The metabolites can be quantified with acceptable precision and accuracy directly from human urine samples after ingestion of 550 mg PABA. The developed LC-MS/MS assay is to our knowledge the first method available for the simultaneous quantification of PABA and its glycine conjugation metabolites in human urine and provides important quantitative data for studies of this phase 2 biotransformation pathway.

  11. Analysis of Protein Expression inBrucella abortusMutants with Different Growth Rates by Two Dimensional Gel Electrophoresis and LC-MS/MS Peptide Analysis. (United States)

    Park, Woo Bin; Im, Young Bin; Soojin, Shim; Yoo, Han Sang


    Brucella abortus is a bacterium that causes brucellosis and is the causative agent of worldwide zoonosis. Pathogenesis of the Brucella abortus infection is so complicates. Thus, several researches have made to solve mechanism in infection of Brucella abortus . While several proteins are revealed as the pathogenic factors by previous researches, underlying mechanism of the Brucella abortus infection is still left to be resolved. We identify the proteins showing the different expression level in Brucella abortus mutants with different biological characteristics that generated by random insertion of transposon. Five mutants were selected based on the biological characteristics, especially the growth features. Total proteins of mutants and wild type were purified and subjected to two dimensional gel electrophoresis. 30 spots of proteins were selected each with an increase and decrease, a change of more than 2-fold was confirmed as compared with the wild type. Selected spots were analyzed using LC-MS/MS peptide analysis. DnaK and ClpB involved in protein aggregation were simultaneously increased. Proteins associated with energy metabolism, SecA and GAPDH were reduced in the expression in some mutants that slower growth rates than wild type. Mutants with slower growth showed decrease with energy metabolism related proteins, while mutants with faster growth showed increase in the pathogenicity related proteins.

  12. Mixtures of quaternary ammonium compounds and anionic organic compounds in the aquatic environment: Elimination and biodegradability in the closed bottle test monitored by LC-MS/MS. (United States)

    Sütterlin, H; Alexy, R; Coker, A; Kümmerer, K


    Quaternary ammonium compounds (QACs) are widely used as disinfectants, detergents and fabric softeners. Anionic detergents are one of the most widely used chemical substances. QACs and anionic surfactants can form ionic pairs. In the present study we investigated the biodegradability of QACs in the presence of different anionic surfactants. The biodegradability of three QACs, namely benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDMAC) and ethacridine lactate (EL), when applied as single substances and in combination with anionic surfactants such as benzene sulfonic acid (BSA), LAS, naphthalene sulfonic acid (NSA) and sodium dodecylsulfonate (SDS) was studied applying the closed bottle test (CBT) [OECD 301D, 1992. Guidelines for Testing of Chemicals. Closed bottle test. Organisation of Economic Cooperation and Development, Paris] at a ratio of 1:1 (mol:mol). Biodegradation was monitored by measuring oxygen concentration in the test vessels with an oxygen electrode in accordance with international standard methods [ISO 5414, 1990. Water quality - determination of dissolved oxygen. In: German Standard Methods for the Examination of Water, Wastewater and Sludge. VCH Verlagsgesellschaft, Weinheim, New York, Basel Cambridge]. Primary elimination of the QACs and of LAS was monitored by LC-MS/MS. There was little biodegradability of the QACs as either single compounds or in the presence of organic counter ions. The biodegradability of the organic counter ions was lower in the presence of QACs as compared to the single substances. Primary elimination of the QACs by sorption took place.

  13. Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics. (United States)

    Burkhart, Julia Maria; Schumbrutzki, Cornelia; Wortelkamp, Stefanie; Sickmann, Albert; Zahedi, René Peiman


    Trypsin is the most frequently used proteolytic enzyme in mass spectrometry-based proteomics. Beside its good availability, it also offers some major advantages such as an optimal average peptide length of ~14 amino acids, and typically the presence of at least two defined positive charges at the N-terminus as well as the C-terminal Arg/Lys, rendering tryptic peptides well suited for CID-based LC-MS/MS. Here, we conducted a systematic study of different types of commercially available trypsin in order to qualitatively and quantitatively compare cleavage specificity, efficiency as well as reproducibility and the potential impact on quantitation and proteome coverage. We present a straightforward strategy applied to complex digests of human platelets, comprising (1) digest controls using a monolithic column HPLC-setup, (2) SCX enrichment of semitryptic/nonspecific peptides, (3) targeted MRM analysis of corresponding full cleavage/missed cleavage peptide pairs as well as (4) LC-MS analyses of complete digests with a three-step data interpretation. Thus, differences in digest performance can be readily assessed, rendering these procedures extremely beneficial to quality control not only the trypsin of choice, but also to effectively compare as well as optimize different digestion conditions and to evaluate the reproducibility of a dedicated digest protocol for all kinds of quantitative proteome studies. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Comparative proteomics of mitosis and meiosis in Saccharomyces cerevisiae. (United States)

    Kumar, Ravinder; Dhali, Snigdha; Srikanth, Rapole; Ghosh, Santanu Kumar; Srivastava, Sanjeeva


    Precise and timely segregation of genetic material and conservation of ploidy are the two foremost requirements for survival of a eukaryotic organism. Two highly regulated cell division processes, namely mitosis and meiosis are central to achieve this objective. The modes of chromosome segregation are distinct in these two processes that generate progeny cells of equal ploidy and half the ploidy in mitosis and meiosis, respectively. Additionally, the nutritional requirement and intracellular processing of biological cue also differ in these two processes. From this, it can be envisaged that proteome of mitotic and meiotic cells will differ significantly. Therefore, identification of proteins that differ in their level of expression between mitosis and meiosis would further reveal the mechanistic detail of these processes. In the present study, we have investigated the protein expression profile of mitosis and meiosis by comparing proteome of budding yeast cultures arrested at mitotic metaphase and metaphase-I of meiosis using proteomic approach. Approximately 1000 and 2000 protein spots were visualized on 2-DE and 2D-DIGE gels respectively, out of which 14 protein spots were significant in 2-DE and 22 in 2D-DIGE (pmitosis, an up-regulation of actin cytoskeleton and its negative regulator occurs in meiosis. Mitosis and meiosis are two different types of cell division cycles with entirely different outcomes with definite biological implication for almost all eukaryotic species. In this work, we investigated, for the first time, the differential proteomic profile of Saccharomyces cerevisiae culture arrested at mitotic metaphase (M) and metaphase-I (MI) of meiosis using 2-DE and 2D-DIGE. Our findings of up-regulation of actin and its negative regulator cofilin during meiosis suggest that the rate of actin cytoskeleton turnover is more in meiosis and actin cytoskeleton may play more crucial role during meiosis compared to mitosis. Present study also suggests that actin

  15. Of mice and men: comparative proteomics of bronchoalveolar fluid. (United States)

    Gharib, S A; Nguyen, E; Altemeier, W A; Shaffer, S A; Doneanu, C E; Goodlett, D R; Schnapp, L M


    We hypothesised that comparing the protein mixture in bronchoalveolar lavage fluid (BALF) between humans and mice may lead to mechanistic insights into common and divergent pathways that evolved in each species. BALF from four humans and six mice was pooled separately and underwent identical shotgun proteomic analysis. Functional and network analysis was applied to identify overlapping and distinct pathways enriched in the BALF. Follow-up experiments using Western analysis in unpooled BALF samples were performed. We identified 91 unique proteins in human and 117 unique proteins in mouse BALF samples. Functional analysis of the proteins revealed conservation of several key processes between the species, including defence response. Oxidative stress response, however, was selectively enriched only in mouse BALF. Differences in the expression of peroxiredoxin-1, a key member of the defence pathway against oxidative injury, were confirmed between normal human and mouse BALF and in models of lung injury. A computational proteomics approach of mouse and human BALF confirms the conservation of immune and defence-mediated pathways while highlighting differences in response to oxidative stress. These observations suggest that the use of mice models to study human lung disorders should be undertaken with an appreciation of interspecies variability.

  16. Comparative Proteomic Profiling of Mycobacterium bovis and BCG Vaccine Strains

    KAUST Repository

    Gao, Ge


    BCG is the only licensed human vaccine currently available against TB. Derived from a virulent strain of M. bovis, the vaccine was thought to have struck a balance between reduced virulence and preserved immunogenicity. Nowadays, BCG vaccine strains used in different countries and vaccination programs show clear variations in their genomes and immune protective properties. The aim of this study was to characterize the proteomic profile on Mycobacterium bovis and five BCG strains Pasteur, Tokyo, Danish, Phipps and Birkhaug by Tandem Mass Tag® (TMT®)-labeling quantitative proteomic approach. In total, 420 proteins were identified and 377 of them were quantitated for their relative abundance. We reported the number and relationship of differential expressed proteins in BCG strains compared to M. bovis and investigated their functions by bioinformatics analysis. Several interesting up-regulated and down-regulated protein targets were found. The identified proteins and their quantitative expression profiles provide a basis for further understanding of the cellular biology of M. bovis and BCG vaccine strains, and hopefully would assist in the design of better anti-TB vaccine and drugs.

  17. Native protein mapping and visualization of protein interactions in the area of human plasma high-density lipoprotein by combining nondenaturing micro 2DE and quantitative LC-MS/MS. (United States)

    Jin, Ya; Bu, Shujie; Zhang, Jun; Yuan, Qi; Manabe, Takashi; Tan, Wen


    A human plasma sample was subjected to nondenaturing micro 2DE and a gel area (5 mm × 18 mm) that includes high-density lipoprotein (HDL) was cut into 1 mm × 1 mm squares, then the proteins in the 90 gel pieces were analyzed by quantitative LC-MS/MS. Grid-cutting of the gel was employed to; (i) ensure the total analysis of the proteins in the area, (ii) standardize the conditions of analysis by LC-MS/MS, (iii) reconstruct the protein distribution patterns from the quantity data. Totally 154 proteins were assigned in the 90 gel pieces and the quantity distribution of each was reconstructed as a color density pattern (a native protein map). The map of apolipoprotein (Apo) A-I showed a wide apparent mass distribution characteristic to HDL and was compared with the maps of the other 153 proteins. Eleven proteins showed maps of wide distribution that overlapped with the map of Apo A-I, and all have been reported to be the components of HDL. Further, seven minor proteins associated with HDL were detected at the gel positions of high Apo A-I quantity. These results for the first time visualized the localization of HDL apolipoproteins on a nondenaturing 2DE gel and strongly suggested their interactions. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Development of a label-free LC-MS/MS strategy to approach the identification of candidate protein biomarkers of disease recurrence in prostate cancer patients in a clinical trial of combined hormone and radiation therapy. (United States)

    Morrissey, Brian; O'Shea, Carmel; Armstrong, John; Rooney, Cathy; Staunton, Lisa; Sheehan, Martina; Shannon, Aoife M; Pennington, Stephen R


    Combined hormone and radiation therapy (CHRT) is one of the principle curative regimes for localised prostate cancer (PCa). Following treatment, many patients subsequently experience disease recurrence however; current diagnostics tests fail to predict the onset of disease recurrence. Biomarkers that address this issue would be of significant advantage. Label-free LC-MS/MS for protein biomarker discovery and MRM for targeted confirmation were applied to patient serum samples accrued in a non-interventional clinical trial of CHRT. Analysis of time-matched patient samples from a patient with disease recurrence compared with a time match disease-free individual supported the identification of 287 proteins. Of these, 141 proteins were quantified, 95 proteins changed in their expression (P ≤ 0.05 and ≥1.5-fold change) and of these 16 were selected for MRM confirmation. The protein expression changes observed in the label-free LC-MS/MS and MRM analysis were found to be highly correlated (R(2) = 0.85). The establishment of a clinical trial to support the acquisition of samples and development of a pipeline for MS-based biomarker discovery and validation should contribute to the identification of a serum protein signature to predict or monitor the outcome of treatment of patients with PCa. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Measurement of Serum and Hepatic Eicosanoids by Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS) in a Mouse Model of Hepatocellular Carcinoma (HCC) with Delivery of c-Met and Activated β-Catenin by Hepatocyte Hydrodynamic Injection. (United States)

    Li, Yanjie; Lin, Nan; Xu, Jianliang; Lu, Yi; Chen, Shuxian; Pan, Chuzhi; Wang, Chusi; Xu, Mingxing; Zhou, Boxuan; Xu, Ruiyun; Xu, Yong-Jiang


    BACKGROUND Most forms of cancer, including hepatocellular carcinoma (HCC), are associated with varying degrees of chronic inflammation. The association between the expression of eicosanoids, which are bioactive lipid mediators of inflammation, and HCC remains unknown. The aim of this study was to measure serum and hepatic eicosanoids in a mouse model of HCC with the delivery of c-Met and activated b-catenin by hepatocyte hydrodynamic injection. MATERIAL AND METHODS The HCC mouse model, and normal control mice, were used in this study with co-delivery of human c-Met combined with activated β-catenin into hepatocytes through hydrodynamic injection. Liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis was used to measure serum and hepatic eicosanoid levels. RESULTS The combined activation of c-Met and β-catenin was induced in the HCC mouse model. LC-MS/MS showed that a total of 13 eicosanoids in serum and 12 eicosanoids in liver tissue were significantly increased in the HCC mice, when compared with control mice. CONCLUSIONS In a mouse model of HCC, co-activation of the c-Met and β-catenin signaling pathway resulted in increased levels of serum and hepatic eicosanoids.

  20. An LC-MS Assay with Isocratic Separation and On-Line Solid Phase Extraction to Improve the Routine Therapeutic Drug Monitoring of Busulfan in Plasma

    Directory of Open Access Journals (Sweden)

    Ialongo Cristiano


    Full Text Available Background: Busulfan (Bu requires therapeutic drug monitoring (TDM in subjects undergoing a conditioning regimen for hematopoietic stem cell transplantation (HSCT. To speed up the procedure and increase reproducibility, we improved our routine LC-MS/MS assay using the on-line solid-phase extraction (SPE of samples.

  1. Detection and differentiation of 22kDa and 20kDa Growth Hormone proteoforms in human plasma by LC-MS/MS

    DEFF Research Database (Denmark)

    Sanmartín, Gerard Such; Bache, N.; Bosch, J.


    a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH...

  2. Enrichment and identification of the most abundant zinc binding proteins in developing barley grains by Zinc-IMAC capture and nano LC-MS/MS

    DEFF Research Database (Denmark)

    Dionisio, Giuseppe; Uddin, Mohammad Nasir; Vincze, Eva


    , and the captured proteins were identified using nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Results: In the endosperm, the most abundant zinc binding proteins were the storage protein B-hordeins, gamma-, and D-hordeins, while in the embryo, 7S globulins storage proteins...

  3. Quantitative antibody-free LC-MS/MS analysis of sTRAIL in sputum and saliva at the sub-ng/mL level

    NARCIS (Netherlands)

    Wilffert, Daniel; Donzelli, Riccardo; Asselman, Angela; Hermans, Jos; Govorukhina, Natalia; ten Hacken, Nick H. T.; Quax, Wim J.; Merbel, van de Nico; Bischoff, Rainer


    Soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL) induces apoptosis via the extrinsic death receptor pathway and maybe a biomarker in the pathogenesis of a broad range of diseases. To investigate the role of sTRAIL in asthma, we developed a quantitative LC-MS/MS method with a

  4. Enrichment and identification of integral membrane proteins from barley aleurone layers by reversed-phase chromatography, SDS-PAGE and LC-MS/MS

    DEFF Research Database (Denmark)

    Hynek, Radovan; Svensson, Birte; Nørregaard Jensen, Ole


    was developed, comprising batch reversed-phase chromatography with stepwise elution of hydrophobic proteins by 2-propanol. Proteins in the most hydrophobic fraction were separated by SDS-PAGE and identified by LC-MS/MS and barley EST sequence database search. The method was efficient for enrichment of integral...

  5. Longitudinal Changes in Circulating Testosterone Levels Determined by LC-MS/MS and by a Commercially Available Radioimmunoassay in Healthy Girls and Boys during the Pubertal Transition

    DEFF Research Database (Denmark)

    Mouritsen, Annette; Søeborg, Tue; Johannsen, Trine Holm


    BACKGROUND: Accurate and selective assessment of testosterone requires use of a sensitive LC-MS/MS method, especially at low levels as those seen in young children. METHODS: The present longitudinal study of 20 healthy children from the Copenhagen Puberty Study followed every 6 months for 5 years...

  6. Validated LC-MS/MS methods for the determination of risperidone and the enantiomers of 9-hydroxyrisperidone in human plasma and urine

    NARCIS (Netherlands)

    De Meulder, Marc; Remmerie, Bart M M; de Vries, Ronald; Sips, Luc L A; Boom, Sandra; Hooijschuur, Edwin W J; van de Merbel, Nico C; Timmerman, Philip M M B L


    Two liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) methods are described, one for the quantitative determination of risperidone and the enantiomers of its active metabolite 9-hydroxyrisperidone (paliperidone) in human plasma and the other for the determination of the

  7. Analysis of sterigmatocystin in cereals, animal feed, seeds, beer and cheese by immunoaffinity column clean-up and HPLC and LC-MS/MS quantification. (United States)

    Marley, Elaine; Brown, Phyllis; Mackie, Jennifer; Donnelly, Carol; Wilcox, Joyce; Pietri, Amedeo; Macdonald, Susan


    A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg(-1) to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg(-1). For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg(-1) the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 and 0.6 µg kg(-1) respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.

  8. A non-targeted approach to chemical discrimination between green tea extract-based dietary supplements and green tea leaves by LC/MS (United States)

    Green tea extract-based dietary supplements (GTDS) have gained in popularity in the U.S. market in recent years. This study evaluated the phytochemical composition of several GTDS in comparison to the composition of green tea leaves using a LC-MS fingerprinting technique coupled with chemometric an...

  9. Reference ranges of 17-hydroxyprogesterone, DHEA, DHEAS, androstenedione, total and free testosterone determined by TurboFlow-LC-MS/MS and associations to health markers in 304 men

    DEFF Research Database (Denmark)

    Jensen, L.S.; Johannsen, T H; Holmboe, Stine Agergaard


    We report reference ranges based on LC-MS/MS for testosterone (T), free testosterone (FT) and its precursors, i.e. 17-hydroxyprogesterone (17-OHP), dehydroepiandrosterone (DHEA), DHEA-sulfate (DHEAS) and androstenedione (Adione), in relation to different health markers and lifestyle factors. The ...

  10. Single Laboratory Validated Method for Determination of Cylindrospermopsin and Anatoxin-a in Ambient Water by Liquid Chromatography/ Tandem Mass Spectrometry (LC/MS/MS) (United States)

    This product is an LC/MS/MS single laboratory validated method for the determination of cylindrospermopsin and anatoxin-a in ambient waters. The product contains step-by-step instructions for sample preparation, analyses, preservation, sample holding time and QC protocols to ensu...

  11. Quantitative Determination of Perfluorochemicals and Fluorotelomer Alcohols in Plants from Biosolid-Amended Fields using LC/MS/MS and GC/MS (United States)

    Analytical methods for determining perfluorochemicals (PFCs) and fluorotelomer alcohols (FTOHs) in plants using liquid chromatography/tandem mass spectrometry (LC/MS/MS) and gas chromatography/mass spectrometry (GC/MS) were developed, and applied to quantify a suite of analytes i...

  12. LC-MS/MS analysis of lipidized analogs of prolactin-releasing peptide utilizing a monolithic column and simple sample preparation

    Czech Academy of Sciences Publication Activity Database

    Zemenová, Jana; Sýkora, D.; Freislebenová, A.; Maletínská, Lenka


    Roč. 9, č. 17 (2017), s. 1319-1328 ISSN 1757-6180 R&D Projects: GA ČR(CZ) GA15-08679S Institutional support: RVO:61388963 Keywords : LC-MS * lipopeptides * monolithic column * prolactin-releasing peptide * stability Subject RIV: CE - Biochemistry OBOR OECD: Biochemical research methods Impact factor: 2.673, year: 2016

  13. Comparative Proteomics of Ovarian Cancer Aggregate Formation Reveals an Increased Expression of Calcium-activated Chloride Channel Regulator 1 (CLCA1). (United States)

    Musrap, Natasha; Tuccitto, Alessandra; Karagiannis, George S; Saraon, Punit; Batruch, Ihor; Diamandis, Eleftherios P


    Ovarian cancer is a lethal gynecological disease that is characterized by peritoneal metastasis and increased resistance to conventional chemotherapies. This increased resistance and the ability to spread is often attributed to the formation of multicellular aggregates or spheroids in the peritoneal cavity, which seed abdominal surfaces and organs. Given that the presence of metastatic implants is a predictor of poor survival, a better understanding of how spheroids form is critical to improving patient outcome, and may result in the identification of novel therapeutic targets. Thus, we attempted to gain insight into the proteomic changes that occur during anchorage-independent cancer cell aggregation. As such, an ovarian cancer cell line, OV-90, was cultured in adherent and non-adherent conditions using stable isotope labeling with amino acids in cell culture (SILAC). Anchorage-dependent cells (OV-90AD) were grown in tissue culture flasks, whereas anchorage-independent cells (OV-90AI) were grown in suspension using the hanging-drop method. Cellular proteins from both conditions were then identified using LC-MS/MS, which resulted in the quantification of 1533 proteins. Of these, 13 and 6 proteins were up-regulated and down-regulated, respectively, in aggregate-forming cells compared with cells grown as monolayers. Relative gene expression and protein expression of candidates were examined in other cell line models of aggregate formation (TOV-112D and ES-2), which revealed an increased expression of calcium-activated chloride channel regulator 1 (CLCA1). Moreover, inhibitor and siRNA transfection studies demonstrated an apparent effect of CLCA1 on cancer cell aggregation. Further elucidation of the role of CLCA1 in the pathogenesis of ovarian cancer is warranted. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Applying quantitative metabolomics based on chemical isotope labeling LC-MS for detecting potential milk adulterant in human milk. (United States)

    Mung, Dorothea; Li, Liang


    There is an increasing demand for donor human milk to feed infants for various reasons including that a mother may be unable to provide sufficient amounts of milk for their child or the milk is considered unsafe for the baby. Selling and buying human milk via the Internet has gained popularity. However, there is a risk of human milk sold containing other adulterants such as animal or plant milk. Analytical tools for rapid detection of adulterants in human milk are needed. We report a quantitative metabolomics method for detecting potential milk adulterants (soy, almond, cow, goat and infant formula milk) in human milk. It is based on the use of a high-performance chemical isotope labeling (CIL) LC-MS platform to profile the metabolome of an unknown milk sample, followed by multivariate or univariate comparison of the resultant metabolomic profile with that of human milk to determine the differences. Using dansylation LC-MS to profile the amine/phenol submetabolome, we could detect an average of 4129 ± 297 (n = 9) soy metabolites, 3080 ± 470 (n = 9) almond metabolites, 4256 ± 136 (n = 18) cow metabolites, 4318 ± 198 (n = 9) goat metabolites, 4444 ± 563 (n = 9) infant formula metabolites, and 4020 ± 375 (n = 30) human metabolites. This high level of coverage allowed us to readily differentiate the six different types of samples. From the analysis of binary mixtures of human milk containing 5, 10, 25, 50 and 75% other type of milk, we demonstrated that this method could be used to detect the presence of as low as 5% adulterant in human milk. We envisage that this method could be applied to detect contaminant or adulterant in other types of food or drinks. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. [Simultaneous determination of daphnetin, daphnoretin, daphneticin in rat plasma by LC-MS/MS and its application in pharmacokinetic study]. (United States)

    Hu, Yi-Yi-Li-Ge-Qi; Cao, Sa-Li; Lin, Long-Fei; Fu, Jing; Dong, Xiao-Xu; Yang, Chun-Jing; Zhang, Miao; Ni, Jian


    To establish HPLC-MS/MS method for simultaneous determination of daphnetin, daphnoretin, and daphneticin in rat plasma after oral and intravenous administration of Daphne giraldii extract, and then use them in the calculation of pharmacokinetic parameters. Six sprague-dawley rats received intragastric administration of D. giraldii extract (daphnetin, daphnoretin and daphneticin were 88.40, 3.24 and 4.28 mg•kg⁻¹, respectively). Their drug plasma concentration was determined by LC-MS/MS with schisandrin as an internal standard to draw plasma concentration-time curve. The pharmacokinetic parameters were calculated by Kinetica 4.4. The results showed that the linear range was 5-1 000 μg•L⁻¹ for daphnetin, daphnoretin and daphneticin, and the method ological test showed conformance to the requirements.The intraday and inter-day variable coefficients (RSD) were both less than 15.0%, indicating that both of legitimate precise and accuracy were consistent with the analysis requirements of biological samples. For daphnetin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 4 h, 858.96 μg•L⁻¹, 10 566.4 μg•L⁻¹•h, 5.19 h and 9.43 h, respectively. For daphnoretin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2.92 h, 178.00 μg•L⁻¹, 905.89 μg•L⁻¹•h, 3.50 h and 6.95 h, respectively. For daphneticin, the pharmacokinetic parameters Tmax, Cmax, AUC0-t, T1/2 and MRT were 2 h, 36.67 μg•L⁻¹, 355.11 μg•L⁻¹•h, 4.95 h and 8.27 h, respectively. The LC-MS/MS analysis method established in this study was proved to be so accurate and sensitive that it can be applied to the pharmacokinetic study of daphnetin, daphnoretin and daphneticin. Copyright© by the Chinese Pharmaceutical Association.

  16. Identifying novel radiotracers for PET imaging of the brain: application of LC-MS/MS to tracer identification. (United States)

    Barth, Vanessa; Need, Anne


    Nuclear medicine imaging biomarker applications are limited by the radiotracers available. Radiotracers enable the measurement of target engagement, or occupancy in relation to plasma exposure. These tracers can also be used as pharmacodynamic biomarkers to demonstrate functional consequences of binding a target. More recently, radiotracers have also been used for patient tailoring in Alzheimer's disease seen with amyloid imaging. Radiotracers for the central nervous system (CNS) are challenging to identify, as they require a unique intersection of multiple properties. Recent advances in tangential technologies, along with the use of iterative learning for the purposes of deriving in silico models, have opened up additional opportunities to identify radiotracers. Mass spectral technologies and in silico modeling have made it possible to measure and predict in vivo characteristics of molecules to indicate potential tracer performance. By analyzing these data alongside other measures, it is possible to delineate guidelines to increase the likelihood of selecting compounds that can perform as radiotracers or serve as the best starting point to develop a radiotracer following additional structural modification. The application of mass spectrometry based technologies is an efficient way to evaluate compounds as tracers in vivo, but more importantly enables the testing of potential tracers that have either no label site or complex labeling chemistry which may deter assessment by traditional means; therefore, use of this technology allows for more rapid iterative learning. The ability to differentially distribute toward target rich tissues versus tissue with no/less target present is a unique defining feature of a tracer. By testing nonlabeled compounds in vivo and analyzing tissue levels by LC-MS/MS, rapid assessment of a compound's ability to differentially distribute in a manner consistent with target expression biology guides the focus of chemistry resources for both

  17. Enantioselective analysis of chloramphenicol residues in honey samples by chiral LC-MS/MS and results of a honey survey. (United States)

    Rimkus, Gerhard G; Hoffmann, Dirk


    Chloramphenicol (CAP) is a broad-spectrum antibiotic used widely in both human and veterinary medication. Since 1994, CAP has not been authorised for use in food-producing animals in the European Union due to several adverse effects. A minimum required performance level (MRPL) of 0.3 µg kg - 1 was established in 2003. The CAP molecule contains two asymmetric centres, thus in total four para-CAP stereoisomers exist. Only the RR-CAP enantiomer is bioactive, having significant antimicrobial activity. For the first time a chiral LC-MS/MS method is reported to identify and quantify the four CAP enantiomers at residue levels in honey samples. The method was validated at two concentration levels. The decision limits (CCα) and detection capabilities (CCß) were well below 0.3 µg kg - 1 , with limits of quantification (LOQs) between 0.08 and 0.12 µg kg - 1 for all four enantiomers. The method provides a sensitive and reliable analysis of CAP enantiomers in honey, and proved its robustness during the daily routine analyses of numerous honey samples. In an internal honey survey, in total 40 honey samples from different geographical regions with identified CAP residues at or above the MRPL were reanalysed by chiral LC-MS/MS. In nine honey samples only the bioactive RR-CAP was detected as anticipated. However, in all other 31 honey samples the non-bioactive SS-CAP was also identified and quantified unambiguously. In 10 of these samples, mixtures of RR- and SS-CAP were analysed, and in 21 samples only the SS-CAP enantiomer, with concentrations up to 2.2 µg kg - 1 . Most of these samples are honeys from Ukraine and Eastern Europe. This is the first report of SS-CAP residues in food samples. The potential sources for these findings are discussed and the need of further systematic studies emphasised. It is recommended to examine in more depth the toxicological profile of the individual CAP stereoisomers.

  18. UV filters analyzed by isotope diluted TurboFlow-LC-MS/MS in urine from Danish children and adolescents. (United States)

    Frederiksen, Hanne; Nielsen, Ole; Skakkebaek, Niels E; Juul, Anders; Andersson, Anna-Maria


    Experimental studies indicate that some chemicals with UV blocking properties (known as UV filters) can act as endocrine disruptors. UV filters are used in sunscreens and other cosmetic- and personal care products, as well as in other consumer products such as food packaging, clothing and furniture textiles to protect the products against UV radiation. Here we present the urinary excretion of suspected endocrine active UV filters in Danish children and adolescents recruited from the general population. The content of benzophenone (BP), benzophenone-1 (BP-1), benzophenone-2 (BP-2), benzophenone-3 (BP-3), 5-chloro-2- hydroxybenzophenone (BP-7), 4-hydroxybenzophenone (4-HBP), 4-methyl-benzophenone (4-MBP), 3-(4- methylbenzylidene)-camphor (4-MBC) and 3-benzylidene camphor (3-BC) were monitored in 24h urine and two consecutive first morning samples from 129 healthy Danish children and adolescents (6-21 yrs). All 387 samples were collected during the autumn (Nov. 2007) and were analyzed by a new on-line TurboFlow-LC-MS/MS method developed for simultaneous biomonitoring of these nine UV filters in urine. BP-3 and BP-1 were detected in more than 80% of the 24h samples and were significantly correlated (R 2 =0.815). BP, 4-HBP and BP-2 were found in 43, 15 and 5% of the samples, respectively. The median (range) concentrations of the UV-filters in 24-h urine were as follows: BP-3, 0.92 (LOD-115); BP-1, 0.54 (LOD-44.6); BP,UV filters; BP-7, 4-MBP, 4-MBC or 3-BC were detected in urine. A highly significant correlation between first morning and 24h urine levels of BP-3, BP-1 and 4-HBP from the same day was observed. Our project on UV filters analyzed by a new robust and sensitive LC-MS/MS method in Danish children and adolescents showed that almost all individuals were exposed to UV filters. Sun protection products have been claimed to be a major source of exposure to sunscreens. However since all children in the present study were exposed during autumn where sunscreens are not

  19. A Conversation on Data Mining Strategies in LC-MS Untargeted Metabolomics: Pre-Processing and Pre-Treatment Steps

    Directory of Open Access Journals (Sweden)

    Fidele Tugizimana


    Full Text Available Untargeted metabolomic studies generate information-rich, high-dimensional, and complex datasets that remain challenging to handle and fully exploit. Despite the remarkable progress in the development of tools and algorithms, the “exhaustive” extraction of information from these metabolomic datasets is still a non-trivial undertaking. A conversation on data mining strategies for a maximal information extraction from metabolomic data is needed. Using a liquid chromatography-mass spectrometry (LC-MS-based untargeted metabolomic dataset, this study explored the influence of collection parameters in the data pre-processing step, scaling and data transformation on the statistical models generated, and feature selection, thereafter. Data obtained in positive mode generated from a LC-MS-based untargeted metabolomic study (sorghum plants responding dynamically to infection by a fungal pathogen were used. Raw data were pre-processed with MarkerLynxTM software (Waters Corporation, Manchester, UK. Here, two parameters were varied: the intensity threshold (50–100 counts and the mass tolerance (0.005–0.01 Da. After the pre-processing, the datasets were imported into SIMCA (Umetrics, Umea, Sweden for more data cleaning and statistical modeling. In addition, different scaling (unit variance, Pareto, etc. and data transformation (log and power methods were explored. The results showed that the pre-processing parameters (or algorithms influence the output dataset with regard to the number of defined features. Furthermore, the study demonstrates that the pre-treatment of data prior to statistical modeling affects the subspace approximation outcome: e.g., the amount of variation in X-data that the model can explain and predict. The pre-processing and pre-treatment steps subsequently influence the number of statistically significant extracted/selected features (variables. Thus, as informed by the results, to maximize the value of untargeted metabolomic data

  20. Validated LC-MS/MS simultaneous assay of five sex steroid/neurosteroid-related sulfates in human serum. (United States)

    Dury, Alain Y; Ke, Yuyong; Gonthier, Renaud; Isabelle, Maxim; Simard, Jean-Nicolas; Labrie, Fernand


    Conventionally, the concentration of steroidal sulfates was estimated by indirect or immuno‑based assays before the use of liquid-chromatography tandem mass spectrometry (LC-MS/MS). In the present study, a validated LC-MS/MS method is described for the simultaneous quantification of dehydroepiandrosterone sulfate (DHEA-S), estrone sulfate (E1‑S), androsterone sulfate (ADT‑S), pregnenolone sulfate (Preg‑S) and allopregnanolone sulfate (Allopreg‑S). E1‑S binding to serum proteins was observed, especially for the high concentration quality control serum samples, leading to -10 to -15% bias using a polymer-based SPE. This protein binding can be efficiently eliminated using a Waters Oasis™ WAX following the same extraction procedure. Most likely, the E1‑S binding elimination on Oasis™ WAX can be attributed to its different sorbent structure, where the benzeno group of E1-S can interact with the benzene of the backbone of Oasis™ WAX. With this improvement, the method has been fully validated according to the FDA guidelines. The low quantification limits (LLOQs) are 40ng/mL, 40pg/mL, 5ng/mL, 1.5ng/mL and 0.25ng/mL for DHEA‑S, E1-S, ADT‑S, Preg‑S and Allopreg-S, respectively. A good linearity is obtained with R>0.99 for all compounds within the appropriate calibration range. Accuracies of all levels of QCs are within the range of 10% for DHEA-S, E1‑S, ADT‑S and Preg‑S while for Allopreg‑S, the accuracy is within the 15% range. The interday coefficient variance is 5.5-9.5% for the low limits of quantification of all five compounds while values of 1.3-9.9% are found for higher levels of QCs of all five compounds. Recovery of the five compounds in stripped serum is equivalent to that in unstripped serum. The average recovery difference is less than 5% between stripped and unstripped serum for each compound. All results of other test parameters such as matrix, hemolysis and lipemic effects as well as stabilities meet the acceptance criteria of

  1. Loss-less Nano-fractionator for High Sensitivity, High Coverage Proteomics

    DEFF Research Database (Denmark)

    Kulak, Nils A; Geyer, Philipp E; Mann, Matthias


    Recent advances in mass spectrometry (MS)-based proteomics now allow very deep coverage of cellular proteomes. To achieve near-comprehensive identification and quantification, the combination of a first HPLC-based peptide fractionation orthogonal to the on-line LC-MS/MS step has proven...

  2. Method validation and analysis of nine dithiocarbamates in fruits and vegetables by LC-MS/MS

    DEFF Research Database (Denmark)

    Schmidt, Bjørn; Christensen, Hanne Bjerre; Petersen, Annette


    for an exposure assessment within the given commodities and a risk assessment by comparing the calculated exposure to the acceptable daily intake and acute reference dose for various exposure groups. The analysis indicated positive findings of DTCs in apples, pears, plums, table grapes, papaya and broccoli...

  3. Phenolic composition of pomegranate peel extracts using an LC-MS approach with silica hydride columns (United States)

    The peels of different pomegranate cultivars (Molla Nepes, Parfianka, Purple Heart, Wonderful and Vkunsyi) were compared in terms of phenolic composition and total phenolics. Analyses were performed on two silica hydride-based stationary phases: phenyl and undecenoic acid columns. Quantitation was ...

  4. Determination of caffeine and paracetamol in Bristol harbour water by LC/MS/MS


    Walter, Y.; Bowdler, P.; Honeychurch, K.


    Anthropogenic inputs such as caffeine and paracetamol can be used as possible chemical markers of sewage pollution as their presence can be presumed to result from human sewage inputs. \\ud In this present investigation we determined the concentrations of caffeine and paracetamol at five sample points within Bristol Floating Harbour, UK and compared these with the levels of Escherichia coli (E. coli) present.

  5. Quantitative analysis of oxytetracycline and its impurities by LC-MS-MS

    DEFF Research Database (Denmark)

    Lykkeberg, Anne Kruse; Halling-Sørensen, Bent; Cornett, Claus


    % and the intraday and interday precision were lower than 7.1%. The method was applied for analysis of commercial available ointments containing OTC resulting in an acceptable quantification of OTC and the impurities in the drug preparations. The advantage of this method compared to the other separation methods...... is an empty separation window right after the large peak corresponding to OTC in the chromatogram, which facilitates an accurate determination of ADOTC and the other impurities....

  6. Postmortem Kanda LC-MS MS ile Yasadışı Madde Taraması 62 Postmortem Adli Olguda Uygulanması

    Directory of Open Access Journals (Sweden)

    Serap Annette Akgür


    Full Text Available Biyolojik örneklerdeki yasadışı madde taraması adli olaylarda büyük bir öneme sahiptir. Kan veya plazma, madde derişim-leri ile bu maddelerin farmakolojik etkileri arasında iyi bir korelasyonun bulunması nedeniyle genellikle tercih edilen materyallerdir. Yasadışı madde analizinde, birçok laboratuar immunoassay yöntemleri ile tarama ve Gaz Kromatografi-Kütle Spektrometresi (GC-MS ile doğrulama şeklinde bir uygulama yapmaktadır. Bununla birlikte, sıvı kromatografi tekniklerinin gelişmesiyle birlikte Sıvı Kromatografisi-Kütle Spektrometresi (LC-MS ve tandem LC-MS (LC-MS/MS birleştirilmiş tekniklerinde özellikle polar ve ısıya dayanıksız olan maddelerin nitel ve nicel analiz amaçlı olarak kullanımı zamanla artmaktadır. Bu çalışmada, LC-ESI (Electrospray İonisation-MS/MS de morfin, (±-amfetamin, (+-metamfetamin, kokain, benzolekgonin, kokaetilen ve (--ll-nor-9-karboksi-t9-THC maddeleri taranmış ve tayin edilmiştir. Sonuç olarak, postmortem kanda uygulanabilecek, türevlendirme işlemine gerek duymayan, duyarlı ve seçimli olarak, çok düşük düzeylerdeki maddelerin doğru ve kesin olarak saptanmasını sağlayacak, LC-MS/MS yöntemi geliştirilmiş ve 62 postmortem adli olguya başarıyla uygulanmıştır. Anahtar kelimeler: Yasadışı madde, tarama, LC/MS/MS, postmortem kan

  7. Comparative Proteomic Analysis of Peritoneal Dialysate from Chronic Glomerulonephritis Patients

    Directory of Open Access Journals (Sweden)

    Hsin-Yi Wu


    Full Text Available Peritoneal dialysis (PD frequently contributes to peritoneal damage which cannot be easily identified without invasive techniques, implying the urgent need for biomarkers and revealing mechanisms. Chronic glomerulonephritis (CGN is one of the leading causes of receiving dialysis treatment. Here, we attempted to analyze the peritoneal dialysate collected from CGN patients when they receive continuous ambulatory peritoneal dialysis (CAPD treatment for the first time and after a year to reveal the protein changes that resulted from PD. Proteins were displayed by two-dimensional gel electrophoresis (2DE. Altered gel spots were digested followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS analysis for protein identification. Eight proteins were found to have differential expression levels between two groups. Their differential expressions were validated by Western blots in other sets of peritoneal dialysates. Proteins identified with higher levels in the first-time dialysate suggested their dominant appearance in CGN patients, while those that showed higher levels in peritoneal dialysate collected after one year may result from initial peritoneal inflammation or changes in the permeability of the peritoneum to middle-sized proteins. All the identified proteins may provide a perceptiveness of peritoneal changes caused by PD and may function as potential biomarkers or drug targets.

  8. Determination of phenolic compounds in Yucca gloriosa bark and root by LC-MS/MS. (United States)

    Montoro, Paola; Skhirtladze, Alexandre; Bassarello, Carla; Perrone, Angela; Kemertelidze, Ether; Pizza, Cosimo; Piacente, Sonia


    On the basis of the biological activities shown by yuccaols and gloriosaols from Yucca schidigera and Yucca gloriosa, the content of yuccaols and gloriosaols in two different parts of Y. gloriosa (roots and bark), was determined for each single compound, and compared with phenolic determination in Y. schidigera bark, concluding that Y. gloriosa bark and roots are rich sources of phenolic derivatives structurally related to resveratrol. LC/ESIMS (liquid chromatography coupled to electrospray mass spectrometry) qualitative and an LC/ESIMS/MS (liquid chromatography coupled to tandem electrospray mass spectrometry) quantitative studies of the phenolic fraction of Y. gloriosa were performed. LC/ESIMS/MS multiple reaction monitoring (MRM) method previously described for yuccaols in Y. schidigera was applied and optimised for separation and determination of gloriosaols and yuccaols in Y. gloriosa. Due to the sensitivity and the repeatability of the assay, we suggest this method as suitable for industrial quality control of raw materials and final products.

  9. Local false discovery rate estimation using feature reliability in LC/MS metabolomics data. (United States)

    Chong, Elizabeth Y; Huang, Yijian; Wu, Hao; Ghasemzadeh, Nima; Uppal, Karan; Quyyumi, Arshed A; Jones, Dean P; Yu, Tianwei


    False discovery rate (FDR) control is an important tool of statistical inference in feature selection. In mass spectrometry-based metabolomics data, features can be measured at different levels of reliability and false features are often detected in untargeted metabolite profiling as chemical and/or bioinformatics noise. The traditional false discovery rate methods treat all features equally, which can cause substantial loss of statistical power to detect differentially expressed features. We propose a reliability index for mass spectrometry-based metabolomics data with repeated measurements, which is quantified using a composite measure. We then present a new method to estimate the local false discovery rate (lfdr) that incorporates feature reliability. In simulations, our proposed method achieved better balance between sensitivity and controlling false discovery, as compared to traditional lfdr estimation. We applied our method to a real metabolomics dataset and were able to detect more differentially expressed metabolites that were biologically meaningful.

  10. Study on the photodegradation of amidosulfuron in aqueous solutions by LC-MS/MS. (United States)

    Benzi, M; Robotti, E; Gianotti, V


    Sulfonylurea herbicides are extensively widespread for the protection of a variety of crops and vegetables because of their low application rates, high selectivity and low persistency in the environment; unfortunately, their low persistence does not always correspond to a lower toxicity, since new species potentially more toxic and stable than the precursor herbicides can form, owing to natural degradation processes. Here, the photodegradation of amidosulfuron in aqueous solutions was studied by high-performance liquid chromatography with diode array detection and tandem mass spectrometry to identify the degradation products in order to outline the environmental fate of the molecules generating from the simulation of one of the natural processes that can occur, i.e., photoinduced degradation. The photodegradation process results in a first order kinetic reaction with a t 1/2 value of 276 h (11.5 days) and a kinetic constant of 0.0027 h(-1), and three possible degradation products were identified. The results obtained are then compared to those obtained in previous works carried out in comparable experimental conditions about nicosulfuron and tribenuron-methyl, two sulfonylurea herbicides belonging to different classes, and to literature data: hypotheses on the existence of preferential degradation pathways are then drawn, in consequence of the molecular structure of the sulfonylurea pesticide. In particular, the use of organic solvents to obtain complete solubilization of the sample plays a fundamental role and deeply influences the degradation processes that, therefore, not always fully adhere to the actual natural photodegradation pathways. Moreover, considerations about toxicity were driven since the complete mineralisation of the sample is not reached: even when the parent pesticides are totally degraded, they are, however, transformed into other organic compounds showing, if subject to ecotoxicological tests, at least the same toxicity of the precursor

  11. Metabolite profiling of ascidian Styela plicata using LC-MS with multivariate statistical analysis and their antitumor activity. (United States)

    Palanisamy, Satheesh Kumar; Trisciuoglio, Daniela; Zwergel, Clemens; Del Bufalo, Donatella; Mai, Antonello


    To identify the metabolite distribution in ascidian, we have applied an integrated liquid chromatography- tandem mass spectrometry (LC-MS) metabolomics approach to explore and identify patterns in chemical diversity of invasive ascidian Styela plicata. A total of 71 metabolites were reported among these alkaloids, fatty acids and lipids are the most dominant chemical group. Multivariate statistical analysis, principal component analysis (PCA) showed a clear separation according to chemical diversity and taxonomic groups. PCA and partial least square discriminant analysis were applied to discriminate the chemical group of S. plicata crude compounds and classify the compounds with unknown biological activities. In this study, we reported for the first time that a partially purified methanol extract prepared from the ascidian S. plicata and Ascidia mentula possess antitumor activity against four tumor cell lines with different tumor histotype, such as HeLa (cervical carcinoma), HT29 (colon carcinoma), MCF-7 (breast carcinoma) and M14 (melanoma). S. plicata fraction SP-50 showed strong inhibition of cell proliferation and induced apoptosis in HeLa and HT29 cells, thus indicating S. plicata fraction SP-50 a potential lead compound for anticancer therapy. The molecular mechanism of action and chemotherapeutic potential of these ascidian unknown biomolecules need further research.

  12. LC/MS/MS analysis of the endogenous dimethyltryptamine hallucinogens, their precursors, and major metabolites in rat pineal gland microdialysate. (United States)

    Barker, Steven A; Borjigin, Jimo; Lomnicka, Izabela; Strassman, Rick


    We report a qualitative liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the simultaneous analysis of the three known N,N-dimethyltryptamine endogenous hallucinogens, their precursors and metabolites, as well as melatonin and its metabolic precursors. The method was characterized using artificial cerebrospinal fluid (aCSF) as the matrix and was subsequently applied to the analysis of rat brain pineal gland-aCSF microdialysate. The method describes the simultaneous analysis of 23 chemically diverse compounds plus a deuterated internal standard by direct injection, requiring no dilution or extraction of the samples. The results demonstrate that this is a simple, sensitive, specific and direct approach to the qualitative analysis of these compounds in this matrix. The protocol also employs stringent MS confirmatory criteria for the detection and confirmation of the compounds examined, including exact mass measurements. The excellent limits of detection and broad scope make it a valuable research tool for examining the endogenous hallucinogen pathways in the central nervous system. We report here, for the first time, the presence of N,N-dimethyltryptamine in pineal gland microdialysate obtained from the rat. Copyright © 2013 John Wiley & Sons, Ltd.

  13. Development and validation of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay to quantify serum voriconazole. (United States)

    Mak, Justin; Sujishi, Kirk K; French, Deborah


    Voriconazole is an azole antifungal drug indicated for use in the treatment of invasive aspergillosis. Due to the large intra- and interindividual variation seen in voriconazole pharmacokinetics along with a high probability of drug-drug interactions, therapeutic drug monitoring is of considerable clinical value. As such, we developed and validated a LC-MS/MS assay to quantify serum voriconazole to improve turnaround time and decrease costs. After protein precipitation with D3-voriconazole (deuterated internal standard) in acetonitrile was performed, samples were separated by gradient elution and injected into the mass spectrometer with a total run-time of 4 min per sample. Multiple reaction monitoring was employed using Q1/Q3 transitions of 350/127 and 350/281 for voriconazole and 353/284 and 353/127 for D3-voriconazole. Sample preparation took 30 min for 6 patient samples. The limit of quantitation was 0.1 μg/mL and the linearity ranged from 0.1 μg/mL to 10.0 μg/mL. Extraction recovery was ∼69% and ion suppression ∼13%. Intra- and inter-assay imprecision (%CV) was voriconazole. Our assay reduces current specimen volume requirements, decreases result turnaround time, and saves institutional funds. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Orthogonal analytical methods for botanical standardization: determination of green tea catechins by qNMR and LC-MS/MS. (United States)

    Napolitano, José G; Gödecke, Tanja; Lankin, David C; Jaki, Birgit U; McAlpine, James B; Chen, Shao-Nong; Pauli, Guido F


    The development of analytical methods for parallel characterization of multiple phytoconstituents is essential to advance the quality control of herbal products. While chemical standardization is commonly carried out by targeted analysis using gas or liquid chromatography-based methods, more universal approaches based on quantitative (1)H NMR (qHNMR) measurements are being used increasingly in the multi-targeted assessment of these complex mixtures. The present study describes the development of a 1D qHNMR-based method for simultaneous identification and quantification of green tea constituents. This approach utilizes computer-assisted (1)H iterative Full Spin Analysis (HiFSA) and enables rapid profiling of seven catechins in commercial green tea extracts. The qHNMR results were cross-validated against quantitative profiles obtained with an orthogonal LC-MS/MS method. The relative strengths and weaknesses of both approaches are discussed, with special emphasis on the role of identical reference standards in qualitative and quantitative analyses. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Environmental contaminants of honeybee products in Uganda detected using LC-MS/MS and GC-ECD.

    Directory of Open Access Journals (Sweden)

    Deborah Ruth Amulen

    Full Text Available Pollinator services and the development of beekeeping as a poverty alleviating tool have gained considerable focus in recent years in sub-Saharan Africa. An improved understanding of the pervasive environmental extent of agro-chemical contaminants is critical to the success of beekeeping development and the production of clean hive products. This study developed and validated a multi-residue method for screening 36 pesticides in honeybees, honey and beeswax using LC-MS/MS and GC-ECD. Of the 36 screened pesticides, 20 were detected. The highest frequencies occurred in beeswax and in samples from apiaries located in the proximity of citrus and tobacco farms. Fungicides were the most prevalent chemical class. Detected insecticides included neonicotinoids, organophosphates, carbamates, organophosphorus, tetrazines and diacylhydrazines. All detected pesticide levels were below maximum residue limits (according to EU regulations and the lethal doses known for honeybees. However, future risk assessment is needed to determine the health effects on the African genotype of honeybees by these pesticide classes and combinations of these. In conclusion, our data present a significant challenge to the burgeoning organic honey sector in Uganda, but to achieve this, there is an urgent need to regulate the contact routes of pesticides into the beehive products. Interestingly, the "zero" detection rate of pesticides in the Mid-Northern zone is a significant indicator of the large potential to promote Ugandan organic honey for the export market.

  16. In Vivo Metabolism Study of Xiamenmycin A in Mouse Plasma by UPLC-QTOF-MS and LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Feng Lei


    Full Text Available Xiamenmycin A is an antifibrotic leading compound with a benzopyran skeleton that is isolated from mangrove-derived Streptomyces xiamenensis. As a promising small molecule for fibrotic diseases, less information is known about its metabolic characteristics in vivo. In this study, the time-course of xiamenmycin A in mouse plasma was investigated by relative quantification. After two types of administration of xiamenmycin A at a single dose of 10 mg/kg, the plasma concentrations were measured quantitatively by LC-MS/MS. The dynamic changes in the xiamenmycin A concentration showed rapid absorption and quick elimination in plasma post-administration. Four metabolites (M1–M4 were identified in blood by UPLC-QTOF-MS, and xiamenmycin B (M3 is the principal metabolite in vivo, as verified by comparison of the authentic standard sample. The structures of other metabolites were identified based on the characteristics of their MS and MS/MS data. The newly identified metabolites are useful for understanding the metabolism of xiamenmycin A in vivo, aiming at the development of an anti-fibrotic drug candidate for the therapeutic treatment of excessive fibrotic diseases.

  17. Rapid and Sensitive Determination of Timosaponin AIII in Rat Plasma by LC-MS/MS and Its Pharmacokinetic Application

    Directory of Open Access Journals (Sweden)

    Zhenzhen Cai


    Full Text Available A rapid sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS method was developed for determination of timosaponin AIII (TA-III in rat plasma, using ginsenoside Re as an internal standard (IS. TA-III and the IS were detected in MRM mode with a negative ionization electrospray mass spectrometer. The calibration curves were linear over the concentration ranges from 11.14 to 1114 ng/mL and the lower limit of quantification (LLOQ was 11.14 ng/mL. Intra-day and inter-day precisions (RSD were within 10%, and accuracy ranged from 6.4% to 9.1%. The extraction recovery at three concentrations ranged from 92.3% to 95.5%. The validated method was successfully applied to monitor the concentrations of TA-III in rat plasma after intragastric administration. The best fit pharmacokinetic model to estimate the pharmacokinetic parameters was a single compartment model with weight of 1/x2 for oral administration groups of rats for TA-III.

  18. Comparison of 7 Published LC-MS/MS Methods for the Simultaneous Measurement of Testosterone, Androstenedione, and Dehydroepiandrosterone in Serum

    DEFF Research Database (Denmark)

    Büttler, Rahel M; Martens, Frans; Fanelli, Flaminia


    , and dehydroepiandrosterone (DHEA). METHODS: We used 7 published LC-MS/MS methods to analyze in duplicate 55 random samples from both men and women. We performed Passing-Bablok regression analysis and calculated Pearson correlation coefficients to assess the agreement of the methods investigated with the median concentration...... measured by all methods, and we calculated the intraassay CV of each method derived from duplicate results and the CVs between the methods. RESULTS: Median concentrations of testosterone were 0.22-1.36 nmol/L for women and 8.27-27.98 nmol/L for men. Androstenedione and DHEA concentrations were 0.......05-5.53 and 0.58-18.04 nmol/L, respectively. Intraassay CVs were 2.9%-10%, 1.2%-8.8%, 2.7%-13%, and 4.3%-16% for testosterone in women, testosterone in men, androstenedione, and DHEA. Slopes of the regression lines calculated by Passing-Bablok regression analysis were 0.92-1.08, 0.92-1.08, 0.90-1.13, and 0...

  19. Mycobiota and Natural Incidence of Aflatoxins, Ochratoxin A, and Citrinin in Indian Spices Confirmed by LC-MS/MS

    Directory of Open Access Journals (Sweden)

    Punam Jeswal


    Full Text Available Nine different Indian spices (red chilli, black pepper, turmeric, coriander, cumin, fennel, caraway, fenugreek, and dry ginger commonly cultivated and highly used in India were analysed for natural occurrence of toxigenic mycoflora and aflatoxins (AFs, ochratoxin A (OTA, and citrinin (CTN contamination. Aspergillus flavus and Aspergillus niger were the most dominant species isolated from all types of spices. Red chilli samples were highly contaminated with aflatoxins (85.4% followed by dry ginger (77.7%. 56% Aspergillus flavus from red chilli and 45% Aspergillus ochraceus from black pepper were toxigenic and produced aflatoxins and ochratoxin A, respectively. Qualitative detection and quantitative detection of mycotoxins in spices were analyzed by ELISA and further confirmed by LC-MS/MS. Penicillium citrinum produced citrinin in red chilli, black pepper, coriander, cumin, fenugreek, and dry ginger samples. The highest amount of AFs was found in red chilli (219.6 ng/g, OTA was in black pepper (154.1 ng/g, and CTN was in dry ginger samples (85.1 ng/g. The results of this study suggest that the spices are susceptible substrate for growth of mycotoxigenic fungi and further mycotoxin production. This is the first report of natural occurrence of citrinin in black pepper and dry ginger from India.

  20. Time Dependency of Chemodiversity and Biosynthetic Pathways: An LC-MS Metabolomic Study of Marine-Sourced Penicillium

    Directory of Open Access Journals (Sweden)

    Catherine Roullier


    Full Text Available This work aimed at studying metabolome variations of marine fungal strains along their growth to highlight the importance of the parameter “time” for new natural products discovery. An untargeted time-scale metabolomic study has been performed on two different marine-derived Penicillium strains. They were cultivated for 18 days and their crude extracts were analyzed by HPLC-DAD-HRMS (High Performance Liquid Chromatography-Diode Array Detector-High Resolution Mass Spectrometry each day. With the example of griseofulvin biosynthesis, a pathway shared by both strains, this work provides a new approach to study biosynthetic pathway regulations, which could be applied to other metabolites and more particularly new ones. Moreover, the results of this study emphasize the interest of such an approach for the discovery of new chemical entities. In particular, at every harvesting time, previously undetected features were observed in the LC-MS (Liquid Chromatography-Mass Spectrometry data. Therefore, harvesting times for metabolite extraction should be performed at different time points to access the hidden metabolome.

  1. Determination of phthalate monoesters in human milk, consumer milk, and infant formula by tandem mass spectrometry (LC-MS-MS)

    DEFF Research Database (Denmark)

    Mortensen, Gerda Krog; Main, Katharina M; Andersson, Anna-Maria


    these phthalates were present, albeit at different concentrations. Median values (microg L(-1)) obtained were 0.11 (mMP), 0.95 (mEP), 3.5 (mBP), 0.8 (mBzP), 9.5 (mEHP), and 101 (mNP). We also analysed seven samples of consumer milk and ten samples of infant formula. Only mBP and mEHP were detected in these samples...... phthalate (mBzP), mono-(2-ethylhexyl) phthalate (mEHP), and monoisononyl phthalate (mNP). The method is based on liquid extraction with a mixture of ethyl acetate and cyclohexane (95:5) followed by two-step solid-phase extraction (SPE). Detection and quantification of the phthalate monoesters were...... accomplished by high-pressure liquid chromatography using a Betasil phenyl column (100 mmx2.1 mmx3 microm) and triple tandem mass spectrometry (LC-MS-MS). Detection limits were in the range 0.01 to 0.5 microg L(-1) and method variation was from 5 to 15%. Analysis of 36 milk samples showed that all...

  2. Environmental contaminants of honeybee products in Uganda detected using LC-MS/MS and GC-ECD. (United States)

    Amulen, Deborah Ruth; Spanoghe, Pieter; Houbraken, Michael; Tamale, Andrew; de Graaf, Dirk C; Cross, Paul; Smagghe, Guy


    Pollinator services and the development of beekeeping as a poverty alleviating tool have gained considerable focus in recent years in sub-Saharan Africa. An improved understanding of the pervasive environmental extent of agro-chemical contaminants is critical to the success of beekeeping development and the production of clean hive products. This study developed and validated a multi-residue method for screening 36 pesticides in honeybees, honey and beeswax using LC-MS/MS and GC-ECD. Of the 36 screened pesticides, 20 were detected. The highest frequencies occurred in beeswax and in samples from apiaries located in the proximity of citrus and tobacco farms. Fungicides were the most prevalent chemical class. Detected insecticides included neonicotinoids, organophosphates, carbamates, organophosphorus, tetrazines and diacylhydrazines. All detected pesticide levels were below maximum residue limits (according to EU regulations) and the lethal doses known for honeybees. However, future risk assessment is needed to determine the health effects on the African genotype of honeybees by these pesticide classes and combinations of these. In conclusion, our data present a significant challenge to the burgeoning organic honey sector in Uganda, but to achieve this, there is an urgent need to regulate the contact routes of pesticides into the beehive products. Interestingly, the "zero" detection rate of pesticides in the Mid-Northern zone is a significant indicator of the large potential to promote Ugandan organic honey for the export market.

  3. Supramolecular separation mechanism of pentafluorophenyl column using ibuprofen and omeprazole as markers: LC-MS and simulation study. (United States)

    Hussain, Afzal; AlAjmi, Mohamed F; Ali, Imran


    The pentafluorophenyl (PFP) column is emerging as a new advancement in separation science to analyze a wide range of analytes and, thus, its separation mechanism at supramolecular level is significant. We developed a mechanism for the separation of ibuprofen and omeprazole using different combinations (ranging from 50:50 to 60:40) of water-acetonitrile containing 0.1% formic acid as the mobile phase. The column used was Waters Acquity UPLC HSS PFP (75 × 2.1 mm, 1.8 μm). The reverse order of elution was observed in different combinations of the mobile phases. The docking study indicated hydrogen bonding between ibuprofen and PFP stationary phase (binding energy was -11.30 kJ/mol). Separation at PFP stationary phase is controlled by hydrogen bonding along with π-π interactions. This stationary phase may be used to analyze both aromatic and aliphatic analytes. The developed mechanism will be useful to separate various analytes by considering the possible interactions, leading to saving of energy, time and money. In addition, this work will be highly useful in preparative chromatography where separation is the major problem at a large scale. Moreover, the developed LC-MS-QTOF method may be used to analyze ibuprofen and omeprazole in an unknown sample owing to the low value of detection limits. Copyright © 2018 John Wiley & Sons, Ltd.

  4. [Determination of tramadol and its active metabolite O-desmethyltramadol in plasma and amniotic fluid using LC/MS/MS]. (United States)

    Zhao, Li-mei; Chen, Xiao-yan; Cui, Jian-jun; Sunita, Maleku; Zhong, Da-fang


    To determinate tramadol and O-desmethyltramadol in human plasma and amniotic fluid by LC/MS/MS, and distribution of tramadol and O-desmethyltramadol in maternity and fetus were studied. Samples containing tramadol, O-desmethyltramadol and diphenhydramine (internal standard, IS ) were extracted using liquid-liquid extraction, followed by liquid chromatographic separation and on-line MS/MS using atmospheric pressure chemical ionization as an interface detection. The analytes were detected in the selected reaction monitoring mode. The calibration curves for tramadol and O-desmethytramadol in plasma and amniotic fluid were linear in the range from 8.0 to 800.0 microg x L(-1) (plasma) and 1.0 to 400.0 microg x L(-1) (amniotic fluid). The method was applied to the measurement of tramadol and O-desmethytramadol concentrations in maternal vein, umbilical vein, umbilical artery and amniotic fluid. Following intramuscular pre-operative administration 1.5 mg x kg(-1) doses of tramadol to parturients, plasma concentrations of tramadol were significantly higher than those in amniotic fluid. The concentrations of O-desmethyltramadol in plasma were lower, and were not detected in amniotic fluid. The method is shown to be accurate, robust and convenient, and suitable for clinical pharmacokinetics studies of tramadol and O-desmethyltramadol.

  5. Statistical modelling coupled with LC-MS analysis to predict human upper intestinal absorption of phytochemical mixtures. (United States)

    Selby-Pham, Sophie N B; Howell, Kate S; Dunshea, Frank R; Ludbey, Joel; Lutz, Adrian; Bennett, Louise


    A diet rich in phytochemicals confers benefits for health by reducing the risk of chronic diseases via regulation of oxidative stress and inflammation (OSI). For optimal protective bio-efficacy, the time required for phytochemicals and their metabolites to reach maximal plasma concentrations (T max ) should be synchronised with the time of increased OSI. A statistical model has been reported to predict T max of individual phytochemicals based on molecular mass and lipophilicity. We report the application of the model for predicting the absorption profile of an uncharacterised phytochemical mixture, herein referred to as the 'functional fingerprint'. First, chemical profiles of phytochemical extracts were acquired using liquid chromatography mass spectrometry (LC-MS), then the molecular features for respective components were used to predict their plasma absorption maximum, based on molecular mass and lipophilicity. This method of 'functional fingerprinting' of plant extracts represents a novel tool for understanding and optimising the health efficacy of plant extracts. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Apple hypanthium firmness: New insights from comparative proteomics

    KAUST Repository

    Marondedze, Claudius


    Fruit firmness constitutes an important textural property and is one of the key parameters for estimating ripening and shelf life, which has a major impact on commercialization. In order to decipher the mechanisms related to firmness of apples (Malus × domestica Borkh.), two-dimensional gel electrophoresis (2-DE) was used to compare the total proteome of high and low firmness phenotypes from apple hypanthia of a \\'Golden Delicious\\' × \\'Dietrich\\' population. A total of 36 differentially regulated protein spots were positively identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and then validated against the Malus expressed sequence tags (EST) database. The findings of this study indicated a lower expression of ethylene biosynthesis related proteins in the high firmness phenotype, which could be linked to the slowing down of the ripening and softening processes. The reduced accumulation of proteins involved in ethylene biosynthesis juxtaposed to the upregulation of a transposase and a GTP-binding protein in the high firmness phenotype. The results also showed higher expression of cytoskeleton proteins in the high firmness phenotype compared to the low firmness phenotype, which play a role in maintaining cell structure and possibly fruit integrity. Finally, a number of proteins involved in detoxification and defense were expressed in fruit hypanthium. This proteomic study provides a contribution towards a better understanding of regulatory networks involved in fruit hypanthium firmness and/or softening, which could be instrumental in the development of improved fruit quality. © 2012 Springer Science+Business Media, LLC.

  7. Quantitative Proteomic Analysis of Serum Proteins from Oral Cancer Patients: Comparison of Two Analytical Methods

    Directory of Open Access Journals (Sweden)

    Yan Yang


    Full Text Available Serum proteomic analysis can be a valuable approach for the discovery of protein biomarkers for early detection or monitoring of a disease. In this study, two analytical methods were compared for quantification of serum proteins in patients with oral cancer. In the first approach, we quantified serum proteins between oral squamous cell carcinoma (OSCC and healthy control subjects by performing in-solution digestion of serum proteins, isobaric tags for relative and absolute quantification (iTRAQ labeling of the resulting peptides, strong cation exchange (SCX fractionation of labeled peptides and finally capillary liquid chromatography with tandem mass spectrometry (LC-MS/MS analysis of the peptides. In the second approach, we first separated serum proteins with SDS-PAGE. The gel-separated proteins were then digested with trypsin and the resulting peptides were labeled with iTRAQ and analyzed with LC-MS/MS for protein quantification. A total of 319 serum proteins were quantified with the first proteomic approach whereas a total of 281 proteins were quantified by the second proteomic approach. Most of the proteins were identified and quantified by both approaches, suggesting that these methods are similarly effective for serum proteome analysis. This study provides compelling evidence that quantitative serum proteomic analysis of OSCC is a valuable approach for identifying differentially expressed proteins in cancer patients’ circulation systems that may be used as potential biomarkers for disease detection. Further validation in large oral cancer patient populations may lead to a simple and low invasive clinical tool for OSCC diagnosis or monitoring.

  8. Improved hydrophilic interaction chromatography LC/MS of heparinoids using a chip with postcolumn makeup flow. (United States)

    Staples, Gregory O; Naimy, Hicham; Yin, Hongfeng; Kileen, Kevin; Kraiczek, Karsten; Costello, Catherine E; Zaia, Joseph


    Heparan sulfate (HS) and heparin are linear, heterogeneous carbohydrates of the glycosaminoglycan (GAG) family that are modified by N-acetylation, N-sulfation, O-sulfation, and uronic acid epimerization. HS interacts with growth factors in the extracellular matrix, thereby modulating signaling pathways that govern cell growth, development, differentiation, proliferation, and adhesion. High-performance liquid chromatography (HPLC)-chip-based hydrophilic interaction liquid chromatography/mass spectrometry has emerged as a method for analyzing the domain structure of GAGs. However, analysis of highly sulfated GAG structures decasaccharide or larger in size has been limited by spray instability in the negative-ion mode. This report demonstrates that addition of postcolumn makeup flow to the amide-HPLC-chip configuration permits robust and reproducible analysis of extended GAG domains (up to degree of polymerization 18) from HS and heparin. This platform provides quantitative information regarding the oligosaccharide profile, degree of sulfation, and nonreducing chain termini. It is expected that this technology will enable quantitative, comparative glycomics profiling of extended GAG oligosaccharide domains of functional interest.

  9. Metabolomic fingerprinting of saffron by LC/MS: novel authenticity markers. (United States)

    Guijarro-Díez, Miguel; Nozal, Leonor; Marina, María Luisa; Crego, Antonio Luis


    An untargeted metabolomic approach using liquid chromatography coupled to electrospray ionization time-of-flight mass spectrometry was developed in this work to identify novel markers for saffron authenticity which is an important matter related to consumer protection, quality assurance, active properties, and also economical impact (saffron is the most expensive spice). Metabolic fingerprinting of authentic and suspicious saffron samples from different geographical origin was obtained and analyzed. Different extracting protocols and chromatographic methodologies were evaluated to obtain the most adequate extracting and separation conditions. Using an ethanol/water mixture at pH 9.0 and an elution gradient with a fused core C18 column enabled obtaining the highest number of significant components between authentic and adulterated saffron. By using multivariate statistical analysis, predictive classification models for authenticity and geographical origin were obtained. Moreover, 84 and 29 significant metabolites were detected as candidates for markers of authenticity and geographical origin, respectively, from which only 34 metabolites were tentatively identified as authenticity markers of saffron, but none related to its geographical origin. Six characteristic compounds of saffron (kaempferol 3-O-glucoside, kaempferol 3-O-sophoroside, kaempferol 3,7-O-diglucoside, kaempferol 3,7,4'-O-triglucoside, kaempferol 3-O-sophoroside-7-O-glucoside, and geranyl-O-glucoside) were confirmed by comparing experimental MS/MS fragmentation patterns with those provided in scientific literature being proposed as novel markers of authenticity. Graphical Abstract Metabolomic fingerprinting of saffron.

  10. A novel microflow LC-MS method for the quantitation of endocannabinoids in serum. (United States)

    Kirkwood, Jay S; Broeckling, Corey D; Donahue, Seth; Prenni, Jessica E


    Endocannabinoids (ECs) represent a class of endogenous, small molecules that bind and activate the G-protein coupled EC receptors. They are involved in a variety of fundamental biological processes and are associated with many disease states. Endocannabinoids are often present in complex matrices and at low concentrations, complicating their measurement. Here we describe a highly sensitive method for the quantitation of the following ECs in serum: N-arachidonoylethanolamine (anandamide), N-oleoylethanolamine, N-palmitoylethanolamine, 2-arachidonoylglycerol, and its inactive isomer 1-arachidonoylglycerol. On-line sample trapping coupled with separation via microflow liquid chromatography and detection by tandem quadrupole mass spectrometry results in the necessary sensitivity for accurate quantitation of ECs in less than 50μL of serum, without the need for off-line solid phase extraction. Limits of quantitation between 1.2 and 13.4pg/mL were achieved, representing a significant increase in sensitivity compared to previous methods using analytical flow rates. An additional benefit of microflow chromatography is the reduction of solvent consumption by more than two orders of magnitude. The experimental utility of the assay is demonstrated through the analysis of serum from hibernating bears to assess seasonal changes in circulating EC concentrations. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. LC/MS/MS identification of some folic acid degradation products after E-beam irradiation

    International Nuclear Information System (INIS)

    Araújo, M.M.; Marchioni, E.; Zhao, M.; Kuntz, F.; Di Pascoli, T.; Villavicencio, A.L.C.H.; Bergaentzle, M.


    Folates belong to the B vitamin group based on the parental compound folic acid (FA). They are involved in important biochemical processes like DNA synthesis and repair. FA is composed of a pteridine ring, p-aminobenzoic acid and glutamate moieties. The human metabolism is not able to synthesize folates and therefore obtain them from diet. FA, a synthetic vitamin, is used as a food fortificant because of its low price, relative stability and increased bioavailability compared to natural folate forms. FA is known to be a sensitive compound easily degradable in aqueous solution by ultraviolet and visible light towards various by-products. Irradiation is a process for preservation of foods that uses accelerated electrons, gamma rays or X-rays. Irradiation is proposed for the treatment of various food products, eliminating or reducing pathogens and insects, increasing the storage time and replacing chemical fumigants. This study concerns the identification of degradation products of FA after E-beam irradiation. FA aqueous solutions were irradiated with a Van de Graaff electrons beam accelerator (2 MeV, 100 μA current, 20 cm scan width, dose rate about 2 kGy/s). Applied doses were between 0 (control) and 10.0 kGy. Absorbed doses were monitored with FWT 60.00 radiochromic dosimeters. - Highlights: ► We investigated the degradation of folic acid aqueous solution after electron beam treatment. ► Radiation doses over 5 kGy promote huge folic acid degradation and appearance of several degradation products. ► PCA, PABA and pABGA, already known folic acid degradation products, are formed due to E-beam treatment. ► Xanthopterin, a new radio-induced breakdown product, is formed after irradiation treatment.

  12. LC-MS/MS Analysis and Pharmacokinetics of Sodium (±-5-Bromo-2-(α-hydroxypentyl Benzoate (BZP, an Innovative Potent Anti-Ischemic Stroke Agent in Rats

    Directory of Open Access Journals (Sweden)

    Xin Tian


    Full Text Available A rapid, sensitive and selective liquid chromatography-triple quadrupole mass spectrometry (LC-MS/MS method was developed and validated for the simultaneous determination of sodium (±-5-Bromo-2-(α-hydroxypentyl benzoate (BZP and its active metabolite 3-butyl-6-bromo-1(3H-isobenzofuranone (Br-NBP in rat plasma using potassium 2-(1-hydroxypentyl-benzoate (PHPB and l-3-n-butylphthalide (NBP as internal standards (IS. Chromatographic separation was achieved on a Hypersil GOLD C18 column using a gradient elution of ammonium acetate and methanol at a flow rate of 0.2 mL/min. Good linearity was achieved within the wide concentration range of 5–10,000 ng/mL. The intra-day and inter-day precision was less than 8.71% and the accuracy was within −8.53% and 6.38% in quality control and the lower limit of quantitation samples. BZP and Br-NBP were stable during the analysis and the storage period. The method was successfully applied to pharmacokinetic studies of BZP in Sprague-Dawley rats for the first time. After a single intravenous administration of BZP at the dose of 0.75 mg/kg, the plasma concentration of BZP and Br-NBP declined rapidly and the AUC0-t of BZP was significantly greater in female rats compared to male rats (p < 0.05. The data presented in this study serve as a firm basis for further investigation of BZP in both preclinical and clinical phases.

  13. Data representing two separate LC-MS methods for detection and quantification of water-soluble and fat-soluble vitamins in tears and blood serum

    Directory of Open Access Journals (Sweden)

    Maryam Khaksari


    Full Text Available Two separate liquid chromatography (LC-mass spectrometry (MS methods were developed for determination and quantification of water-soluble and fat-soluble vitamins in human tear and blood serum samples. The water-soluble vitamin method was originally developed to detect vitamins B1, B2, B3 (nicotinamide, B5, B6 (pyridoxine, B7, B9 and B12 while the fat-soluble vitamin method detected vitamins A, D3, 25(OHD3, E and K1. These methods were then validated with tear and blood serum samples. In this data in brief article, we provide details on the two LC-MS methods development, methods sensitivity, as well as precision and accuracy for determination of vitamins in human tears and blood serum. These methods were then used to determine the vitamin concentrations in infant and parent samples under a clinical study which were reported in "Determination of Water-Soluble and Fat-Soluble Vitamins in Tears and Blood Serum of Infants and Parents by Liquid Chromatography/Mass Spectrometry DOI:10.1016/j.exer.2016.12.007 [1]". This article provides more details on comparison of vitamin concentrations in the samples with the ranges reported in the literature along with the medically accepted normal ranges. The details on concentrations below the limits of detection (LOD and limits of quantification (LOQ are also discussed. Vitamin concentrations were also compared and cross-correlated with clinical data and nutritional information. Significant differences and strongly correlated data were reported in [1]. This article provides comprehensive details on the data with slight differences or slight correlations.

  14. Simultaneous determination of vitamins A and D3 in dairy products by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (United States)

    Barakat, I. S. A.; Hammouri, M. K.; Habib, I.


    A potential method for simultaneous determination of vitamin A and vitamin D3 (25- hydroxyvitamin D3) in fresh milk samples is addressed. The method is based on combination of high performance liquid chromatography and mass spectrometry during the course of analysis. The method applied for determination of vitamins A and D3 on eighteen (18) different fresh milk samples using liquid chromatography along with tandem -mass spectrometry. The work describes the suitability of the proposed method for the simultaneous determination of both vitamins using LC-MS/MS as a specific and quantitative technique. The vitamins of milk were separated by C18 Thermo gold column(100mm × 4.6mm × 5 μm) with a flow rate of 1ml/min (using an isocratic mobile phase). The method was validated using duplicate analyses, relative recovery experiment, and comparative analysis with control samples. Liquid- liquid extraction was employed as a pre-concentration step with n-hexane - dichloromethane mixture (90%:10%) as an extraction solvent. The molecular ions (m/z) appeared near 286 and 385nm and for the base peaks were appeared near 255 and 355nm for vitamins A and D3. Good correlation coefficients were obtained, 0.9999 for vitamin D3 and 0.9994 for vitamin A. The limit of detection and the limit of quantification were found to be 0.09ng/ml and 0.54ng/ml for vitamin D3 and 0.32ng/ml and 1.8ng/ml and for vitamin A. The proposed method showed excellent recoveries, about 98% for both vitamins A and D3.

  15. Applications of monolithic silica capillary columns in proteomics

    NARCIS (Netherlands)

    Barroso, B.; Lubda, D; Bischoff, Rainer


    The use and applicability of silica based capillary monolithic reversed-phase columns in proteomic analysis has been evaluated by liquid chromatography-mass spectrometry (LC-MS). Chromatographic performance of the monolithic capillaries was evaluated with a tryptic digest of cytochrome C showing

  16. Proteomic profiling of an undefined microbial consortium cultured in fermented dairy manure: Methods development. (United States)

    Hanson, Andrea J; Paszczynski, Andrzej J; Coats, Erik R


    The production of polyhydroxyalkanoates (PHA; bioplastics) from waste or surplus feedstocks using mixed microbial consortia (MMC) and aerobic dynamic feeding (ADF) is a growing field within mixed culture biotechnology. This study aimed to optimize a 2DE workflow to investigate the proteome dynamics of an MMC synthesizing PHA from fermented dairy manure. To mitigate the challenges posed to effective 2DE by this complex sample matrix, the bacterial biomass was purified using Accudenz gradient centrifugation (AGC) before protein extraction. The optimized 2DE method yielded high-quality gels suitable for quantitative comparative analysis and subsequent protein identification by LC-MS/MS. The optimized 2DE method could be adapted to other proteomic investigations involving MMC in complex organic or environmental matrices. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Proteome of Salmonella Enterica SerotypeTyphimurium Grown in a Low Mg2+/pH Medium

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Liang; Ansong, Charles; Smallwood, Heather S.; Rommereim, Leah M.; McDermott, Jason E.; Brewer, Heather M.; Norbeck, Angela D.; Taylor, Ronald C.; Gustin, Jean K.; Heffron, Fred; Smith, Richard D.; Adkins, Joshua N.


    The facultative intracellular pathogen Salmonella enterica serovar Typhimurium (STM) must replicate within host macrophages in order to establish systemic infection in susceptible mice. In an effort to identify new STM proteins that help the bacterium colonize macrophages, we have cultured STM cells with a low pH/low magnesium medium (MgM) under two different conditions termed MgM-Shock and MgM-Dilution and investigated the impacts of these culturing conditions on the STM proteome by using liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics. LC-MS/MS results showed that alteration of culturing conditions affected a group of STM proteins differently. Compared to MgM-Shock, MgM-Dilution induced more proteins of the Salmonella-pathogenecity island 2-type III secretion system (SPI2-T3SS). The abundances of the proteins used for cobalamin biosynthesis increased under MgM-Shock condition but decreased under MgM-Dilution condition, while those proteins used for thiamine or biotin biosynthesis were not affected under the former condition but increased under the latter condition. Western-blot (WB) analysis confirmed the LC-MS/MS results. Because cobalamin, thiamine and biotin play different roles in STM metabolism, differential induction of the proteins involved in their biosyntheses suggests that the metabolic states of STM cells under these conditions differ considerably. WB analysis also showed that the abundances of SPI2-T3SS proteins SsaQ and SseE and biotin biosynthesis proteins BioB and BioD increased after STM infection of RAW 264.7 macrophages. Deletion of the gene encoding BioB reduced the ability of STM to replicate inside the macrophages, demonstrating for the first time the involvement of a biotin synthesis protein in STM colonization of macrophages.

  18. Multiresidue determination and potential risks of emerging pesticides in aquatic products from Northeast China by LC-MS/MS. (United States)

    Fu, Lei; Lu, Xianbo; Tan, Jun; Wang, Longxing; Chen, Jiping


    A simple method for determining 33 pesticides with a wide polarity range (logK ow 0.6-4.5) in aquatic products was developed based on LC-MS/MS. The target analytes included three types of widely used pesticides: insecticides, fungicides and herbicides. Based on the optimization of ultrasonic assisted extraction and GPC clean-up procedures, the matrix effect, extraction recoveries and LOD were improved distinctively. LOQ of this method was below 0.5ng/g for all pesticides, which is superior to values in the literature, and the matrix effect was reduced effectively (-14.7% to 7.5%). The method was successfully applied to investigate the pesticide residue levels of twenty-five samples including seven common kinds of fishes from Northeast China. The results showed that all targeted pesticides were present in the fish samples; however, their levels were low, except for atrazine, linuron, ethoprophos, tetrachlorvinphos, acetochlor and fenthion. Atrazine and linuron caught our attention because the concentrations of atrazine in fish samples from Liaoning province were in the range of 0.5-8ng/g (w/w) with mean concentration of 2.3ng/g, which were far above those of other pesticides. The levels of linuron were in the range of 0.6-6ng/g (mean concentration 2.8ng/g), which were the highest among all targeted pesticides in the Inner Mongolia. This is the first systematic investigation on the characteristics and levels of these pesticides in aquatic products from northeast China. Considering their toxicity and bioaccumulation, the potential risk of atrazine and linuron from consuming aquatic products should be paid more attention. Copyright © 2017. Published by Elsevier B.V.

  19. Dissipation kinetics and degradation mechanism of amicarbazone in soil revealed by a reliable LC-MS/MS method. (United States)

    Dong, Maofeng; Han, Wei; Ediage, Emmanuel Njumbe; Fan, Liangxiu; Tang, Hongxia; Wang, Weimin; Han, Lijun; Zhao, Zhihui; Song, Weiguo; Han, Zheng


    A sensitive and reliable analytical method was developed for simultaneous determination of amicarbazone (AMZ) and its two major metabolites including desamino amicarbazone (DA) and isopropyl-2-hydroxy-DA-amicarbazone (Ipr-2-OH-DA-AMZ) in soil for the first time. Targeted analytes were extracted and purified using a modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) procedure, and then analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a total run time of 9 min. The established approach was extensively validated by determining the linearity (R (2) ≥ 0.99), recovery (84-96 ), sensitivity (limits of quantification at 5-10 μg kg(-1)), and precision (RSDs ≤12 %). Based on the methodological advances, the subsequent dissipation kinetics and degradation mechanism of amicarbazone in soil were thoroughly investigated in an illumination incubator. As revealed, AMZ was easily degraded with the half-lives of 13.9-19.7 days in soil. Field trial results of AMZ (40 g a.i. ha(-1)) in Shanghai showed that the residues of AMZ and its metabolite Ipr-2-OH-DA-AMZ decreased from 0.505 mg kg(-1) (day 50) to 0.038 mg kg(-1) (day 365) and from 0.099 mg kg(-1) (day 50) to 0.028 mg kg(-1) (day 365), respectively, while the content of DA increased from 0.097 mg kg(-1) (day 50) to 0.245 mg kg(-1) (day 365). This study provided valuable data to understand the toxicity of AMZ and substantially promote its safe application to protect environment and human health.

  20. A novel LC-MS/MS method for the simultaneous quantification of topiramate and its main metabolites in human plasma. (United States)

    Milosheska, Daniela; Roškar, Robert


    The aim of the present report was to develop and validate simple, sensitive and reliable LC-MS/MS method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM, 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and afterwards used as an authentic standard for method development and validation. Sample preparation method employs 100μL of plasma sample and liquid-liquid extraction with a mixture of ethyl acetate and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using Kinetex C18 column (50×2.1mm, 2.6μm) by gradient elution using water and methanol as a mobile phase and stable isotope labeled TPM as internal standard. The method showed to be selective, accurate, precise and linear over the concentration ranges of 0.10-20μg/mL for TPM, 0.01-2.0μg/mL for 2,3-desisopropylidene TPM, and 0.001-0.200μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM. The described method is the first fully validated method capable of simultaneous determination of TPM and its main metabolites in plasma over the selected analytical range. The suitability of the method was successfully demonstrated by the quantification of all analytes in plasma samples of patients with epilepsy and can be considered as reliable analytical tool for future investigations of the TPM metabolism. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Simultaneous analysis of regorafenib and sorafenib and three of their metabolites in human plasma using LC-MS/MS. (United States)

    Allard, Marie; Khoudour, Nihel; Rousseau, Benoît; Joly, Charlotte; Costentin, Charlotte; Blanchet, Benoît; Tournigand, Christophe; Hulin, Anne


    A new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, performed by electrospray ionization in positive mode using a triple quadrupole mass spectrometry, has been developed and validated for the simultaneous determination of regorafenib (REGO), its two metabolites regorafenib-M2 and regorafenib-M5, sorafenib (SORA), and its N-oxide metabolite in human plasma. Separation is achieved on an Hypersil Gold ® column using a gradient elution of 10mM ammonium formate containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) at a flow rate of 0.3mL/min. After addition of two internal standards and a protein precipitation, the supernatant is diluted two-fold in a 0.1% (v/v) formic acid solution. Two selected reaction monitoring transitions are used, for each analyte, one for quantitation and the second one for confirmation. The standard curves are ranged from 50 to 5 000ng/mL for REGO and its metabolites and 80 to 5 000ng/mL for SORA and its metabolite and were fitted to a 1/x weighted linear regression model. The method also showed satisfactory results in terms of sensitivity, specificity, precision (intra- and inter-day CV from 2.4 to 10.2%), accuracy (from 91.0 to 111.7%), recovery as well as stability of the analytes under various conditions. The method is usually used in clinical practice in order to improve the SORA treatment for renal carcinoma, REGO treatment for colorectal cancer and both for hepatocellular carcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Determination of tacrine-6-ferulic acid in rat plasma by LC-MS/MS and its application to pharmacokinetics study. (United States)

    Sun, Xiuman; Zhang, Peiting; Pi, Rongbiao; Zhou, Yuting; Deng, Xuejiao; Xie, Zhiyong; Liao, Qiongfeng


    Tacrine, as a drug for treating Alzheimer's disease (AD), has low efficacy owing to its single function and serious side effects. However, tacrine-6-ferulic acid (T6FA), the dimer which added ferulic acid to tacrine, has been found to be a promising multifunctional drug candidate for AD and much more potent and selective on acetylcholinesterase (AChE) than tacrine. The aim of the present work was to develop and validate an LC-MS/MS method with electrospray ionization for the quantification of T6FA in rat plasma using tacrine-3-ferulic acid (T3FA) as internal standard and to examine its application for pharmacokinetic study in rats. Following a single liquid-liquid extraction with ethyl acetate, chromatographic separation was achieved at 25 °C on a BDS Hypersil C18 column with a mobile phase composed of 1% formic acid and methonal (30:70, v/v) at a flow rate of 0.2 mL/min. Quantification was achieved by monitoring the selected ions at m/z 474.2 → 298.1 for T6FA and m/z 432.2 → 199.0 for T3FA. The method was validated to be rapid, specific, accurate and precise over the concentration range of 0.5-1000.0 ng/mL in rat samples. Furthermore, it was successfully applied for the pharmacokinetic measurement of T6FA with an oral administration at 40 mg/kg to rats. Copyright © 2014 John Wiley & Sons, Ltd.

  3. An optimized and validated SPE-LC-MS/MS method for the determination of caffeine and paraxanthine in hair. (United States)

    De Kesel, Pieter M M; Lambert, Willy E; Stove, Christophe P


    Caffeine is the probe drug of choice to assess the phenotype of the drug metabolizing enzyme CYP1A2. Typically, molar concentration ratios of paraxanthine, caffeine's major metabolite, to its precursor are determined in plasma following administration of a caffeine test dose. The aim of this study was to develop and validate an LC-MS/MS method for the determination of caffeine and paraxanthine in hair. The different steps of a hair extraction procedure were thoroughly optimized. Following a three-step decontamination procedure, caffeine and paraxanthine were extracted from 20 mg of ground hair using a solution of protease type VIII in Tris buffer (pH 7.5). Resulting hair extracts were cleaned up on Strata-X™ SPE cartridges. All samples were analyzed on a Waters Acquity UPLC® system coupled to an AB SCIEX API 4000™ triple quadrupole mass spectrometer. The final method was fully validated based on international guidelines. Linear calibration lines for caffeine and paraxanthine ranged from 20 to 500 pg/mg. Precision (%RSD) and accuracy (%bias) were below 12% and 7%, respectively. The isotopically labeled internal standards compensated for the ion suppression observed for both compounds. Relative matrix effects were below 15%RSD. The recovery of the sample preparation procedure was high (>85%) and reproducible. Caffeine and paraxanthine were stable in hair for at least 644 days. The effect of the hair decontamination procedure was evaluated as well. Finally, the applicability of the developed procedure was demonstrated by determining caffeine and paraxanthine concentrations in hair samples of ten healthy volunteers. The optimized and validated method for determination of caffeine and paraxanthine in hair proved to be reliable and may serve to evaluate the potential of hair analysis for CYP1A2 phenotyping. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Quantitative determination of metaxalone in human plasma by LC-MS and its application in a pharmacokinetic study

    Directory of Open Access Journals (Sweden)

    Lanting Zhao


    Full Text Available A simple and rapid method using liquid chromatography–mass spectrometry (LC-MS for the determination of metaxalone in human plasma has been developed and validated. Letrozole was used as the internal standard (IS. The plasma samples were simply treated with acetonitrile which allowed the precipitation of plasma proteins. The chromatographic separation was achieved on a Sapphire C18 (2.1 mm × 150 mm, 5 µm, Newark, USA column using the mobile phase (5 mM ammonium acetate containing 0.01% formic acid: acetonitrile (45:55, v/v at a flow rate of 0.3 ml/min. The selected ion monitoring (SIM in the positive mode was used for the determination of [M + H]+ m/z 222.1 and 286.1 for metaxalone and letrozole, respectively. The standard curve obtained was linear (r2 ≥ 0.99 over the concentration range of 30.24−5040 ng/ml. Meanwhile, no interfering peaks or matrix effect was observed. The method established was simple and successfully applied to a pharmacokinetic study of metaxalone in healthy Chinese volunteers after a single oral dose administration of 800 mg metaxalone. The main pharmacokinetic parameters of metaxalone were as follow: Cmax, (1664 ± 1208 ng/ml and (2063 ± 907 ng/ml; AUC0−36, (13925 ± 6590 ng/ml h and (18620 ± 5717 ng/ml h; t1/2, (13.6 ± 7.7 h and (20.3 ± 7.7 h for the reference and test tablets, respectively. These pharmacokinetic parameters of metaxalone in healthy Chinese volunteers were reported for the first time.

  5. [Determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials by LC-MS/MS]. (United States)

    Si, Hai-hong; Li, Yan-jing; Xue, Jia; Huang, Wen-zhe; Wang, Zhen-zhong; Xiao, Wei


    To develop a LC-MS/MS method for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials, the column was Agilent ZORBAX Eclipse plus C18 (3.0 mm x 50 mm, 1.8 µm), and the mobile phase consisted of methanol-water (containing 0.2% formic acid) (95:5) at a flow rate of 0.5 mL · min(-1). The multiple reaction ion monitoring (MRM) with an ESI interface in the negative ion mode was selected. The results showed that the linear ranges of five kinds of ginkgolic acids were in the range of 0.2-36.0 µg · L(-1) (r ≥ 0.999 5). The lowest limit of quantification (LOQ) of ginkgo acid C13: 0, C15:1, C17:2, C15:0 and C17:1 were 0.18, 0.18, 0.21, 0.10 and 0.20 µg · L(-1), respectively. The average recovery was between 73.28% and 87.56%, and the average content of total ginkgolic acids in three batches of samples was in the range of 0.023-0.028 µg · g(-1), which was much lower than 2 µg · g(-1) prescribed in drug registration standards. This method is simple and rapid with high sensitivity, which can be used for the determination of five kinds of trace ginkgolic acids in diterpene ginkgolides meglumine injection materials.

  6. Comparative proteomic analysis of ductal and lobular invasive breast carcinoma. (United States)

    Oliveira, N C S; Gomig, T H B; Milioli, H H; Cordeiro, F; Costa, G G; Urban, C A; Lima, R S; Cavalli, I J; Ribeiro, E M S F


    Breast cancer is the second most common cancer worldwide and the first among women. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two major histological subtypes, and the clinical and molecular differences between them justify the search for new markers to distinguish them. As proteomic analysis allows for a powerful and analytical approach to identify potential biomarkers, we performed a comparative analysis of IDC and ILC samples by using two-dimensional electrophoresis and mass spectrometry. Twenty-three spots were identified corresponding to 10 proteins differentially expressed between the two subtypes. ACTB, ACTG, TPM3, TBA1A, TBA1B, VIME, TPIS, PDIA3, PDIA6, and VTDB were upregulated in ductal carcinoma compared to in lobular carcinoma samples. Overall, these 10 proteins have a key role in oncogenesis. Their specific functions and relevance in cancer initiation and progression are further discussed in this study. The identified peptides represent promising biomarkers for the differentiation of ductal and lobular breast cancer subtypes, and for future interventions based on tailored therapy.

  7. Identification of azurocidin as a potential periodontitis biomarker by a proteomic analysis of gingival crevicular fluid

    Directory of Open Access Journals (Sweden)

    Lee Jae-Mok


    Full Text Available Abstract Background The inflammatory disease periodontitis results in tooth loss and can even lead to diseases of the whole body if not treated. Gingival crevicular fluid (GCF reflects the condition of the gingiva and contains proteins transuded from serum or cells at inflamed sites. In this study, we aimed to discover potential protein biomarkers for periodontitis in GCF proteome using LC-MS/MS. Results We identified 305 proteins from GCF of healthy individuals and periodontitis patients collected using a sterile gel loading tip by ESI-MS/MS coupled to nano-LC. Among these proteins, about 45 proteins were differentially expressed in the GCF proteome of moderate periodontitis patients when compared to the healthy individuals. We first identified azurocidin in the GCF, but not the saliva, as an upregulated protein in the periodontitis patients and verified its increased expression during periodontitis by ELISA using the GCF of the classified periodontitis patients compared to the healthy individuals. In addition, we found that azurocidin inhibited the differentiation of bone marrow-derived macrophages to osteoclasts. Conclusions Our results show that GCF collection using a gel loading tip and subsequent LC-MS/MS analysis following 1D-PAGE proteomic separation are effective for the analysis of the GCF proteome. Our current results also suggest that azurocidin could be a potential biomarker candidate for the early detection of inflammatory periodontal destruction by gingivitis and some chronic periodontitis. Our data also suggest that azurocidin may have an inhibitory role in osteoclast differentiation and, thus, a protective role in alveolar bone loss during the early stages of periodontitis.

  8. Combination of LC-MS2 and GC-MS as a Tool to Differentiate Oxidative Metabolites of Zearalenone with Different Chemical Structures

    Directory of Open Access Journals (Sweden)

    Andreas A. Hildebrand


    Full Text Available Recent studies on the mammalian and fungal metabolism of the mycotoxin zearalenone (ZEN have disclosed the formation of six regioisomers of monohydroxy-ZEN and its reductive metabolite zearalenol (ZEL. Hydroxylation occurs at the aromatic ring or at one of four positions of the aliphatic macrocycle. In addition, an aliphatic ZEN epoxide, its hydrolysis product, and other products were identified in fungal cultures. In this paper, we report the product ion spectra of the [M-H]− ions of 22 oxidative metabolites of ZEN and ZEL, obtained by LC-MS2 analysis using a linear ion trap mass spectrometer with negative electrospray ionization. The MS2 spectra exhibit qualitative and quantitative differences which allow a clear distinction of most metabolites. Moreover, GC-MS analysis of the trimethylsilylated metabolites yields electron impact mass spectra with numerous fragment ions which can be used as fingerprint to confirm the chemical structure derived by LC-MS2 analysis.

  9. Quantification of in vivo site-specific Asp isomerization and Asn deamidation of mAbs in animal serum using IP-LC-MS. (United States)

    Mehl, John T; Sleczka, Bogdan G; Ciccimaro, Eugene F; Kozhich, Alexander T; Gilbertson, Deb G; Vuppugalla, Ragini; Huang, Christine S; Stevens, Brenda; Mo, Jingjie; Deyanova, Ekaterina G; Wang, Yun; Huang, Richard Yc; Chen, Guodong; Olah, Timothy V


    Isomerization of aspartic acid and deamidation of asparagine are two common amino acid modifications that are of particular concern if located within the complementarity-determining region of therapeutic antibodies. Questions arise as to the extent of modification occurring in circulation due to potential exposure of the therapeutic antibody to different pH regimes. To enable evaluation of site-specific isomerization and deamidation of human mAbs in vivo, immunoprecipitation (IP) has been combined with LC-MS providing selective enrichment, separation and detection of naive and modified forms of tryptic peptides comprising complementarity-determining region sequences. IP-LC-MS can be applied to simultaneously quantify in vivo drug concentrations and measure the extent of isomerization or deamidation in PK studies conducted during the drug discovery stage.

  10. Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays

    DEFF Research Database (Denmark)

    Weisser, Johan Juhl; Hansen, Cecilie Hurup; Poulsen, Rikke


    Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination...... of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis....... The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off...

  11. Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides. (United States)

    Ciccimaro, Eugene; Ranasinghe, Asoka; D'Arienzo, Celia; Xu, Carrie; Onorato, Joelle; Drexler, Dieter M; Josephs, Jonathan L; Poss, Michael; Olah, Timothy


    Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte's surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte's unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma.

  12. Antibody-drug conjugate bioanalysis using LB-LC-MS/MS hybrid assays: strategies, methodology and correlation to ligand-binding assays. (United States)

    Wang, Jian; Gu, Huidong; Liu, Ang; Kozhich, Alexander; Rangan, Vangipuram; Myler, Heather; Luo, Linlin; Wong, Richard; Sun, Huadong; Wang, Bonnie; Vezina, Heather E; Deshpande, Shrikant; Zhang, Yan; Yang, Zheng; Olah, Timothy V; Aubry, Anne-Francoise; Arnold, Mark E; Pillutla, Renuka; DeSilva, Binodh


    Antibody-drug conjugates (ADCs) are complex drug constructs with multiple species in the heterogeneous mixture that contribute to their efficacy and toxicity. The bioanalysis of ADCs involves multiple assays and analytical platforms. A series of ligand binding and LC-MS/MS (LB-LC-MS/MS) hybrid assays, through different combinations of anti-idiotype (anti-Id), anti-payload, or generic capture reagents, and cathepsin-B or trypsin enzyme digestion, were developed and evaluated for the analysis of conjugated-payload as well as for species traditionally measured by ligand-binding assays, total-antibody and conjugated-antibody. Hybrid assays are complementary or viable alternatives to ligand-binding assay for ADC bioanalysis and PK/PD modeling. The fit-for-purpose choice of analytes, assays and platforms and an integrated strategy from Discovery to Development for ADC PK and bioanalysis are recommended.

  13. Comparative proteomic study on Brassica hexaploid and its parents provides new insights into the effects of polyploidization. (United States)

    Shen, Yanyue; Zhang, Yu; Zou, Jun; Meng, Jinling; Wang, Jianbo


    Polyploidy has played an important role in promoting plant evolution through genomic merging and doubling. Although genomic and transcriptomic changes have been observed in polyploids, the effects of polyploidization on proteomic divergence are poorly understood. In this study, we reported quantitative analysis of proteomic changes in leaves of Brassica hexaploid and its parents using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with mass spectrometry. A total of 2044 reproducible proteins were quantified by at least two unique peptides. We detected 452 proteins differentially expressed between Brassica hexaploid and its parents, and 100 proteins were non-additively expressed in Brassica hexaploid, which suggested a trend of non-additive protein regulation following genomic merger and doubling. Functional categories of cellular component biogenesis, immune system process, and response to stimulus, were significantly enriched in non-additive proteins, probably providing a driving force for variation and adaptation in allopolyploids. In particular, majority of the total 452 differentially expressed proteins showed expression level dominance of one parental expression, and there was an expression level dominance bias toward the tetraploid progenitor. In addition, the percentage of differentially expressed proteins that matched previously reported differentially genes were relatively low. This study aimed to get new insights into the effects of polyploidization on proteomic divergence. Using iTRAQ LC-MS/MS technology, we identified 452 differentially expressed proteins between allopolyploid and its parents which involved in response to stimulus, multi-organism process, and immune system process, much more than previous studies using 2-DE coupled with mass spectrometry technology. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in allopolyploid and its parents, which will lead to a better understanding of

  14. Validated LC-MS/MS Method for the Quantification of Free and Bound Lignans in Cereal-Based Diets and Feces

    DEFF Research Database (Denmark)

    Nørskov, Natalja; Knudsen, Knud Erik Bach


    lignans (matairesinol, hydroxymatairesinol, secoisolariciresinol, lariciresinol, isolariciresinol, syringaresinol, medioresinol, and pinoresinol) and two enterolignans (enterodiol and enterolactone) in cereal-based diets/bread and feces. The method consisted of alkaline methanolic extraction combined......Despite the extensive literature describing the biological effects of phenolic compounds from cereals, little is known about their bioaccessibility in the food matrix. This paper describes a validated LC-MS/MS method for the quantification of free and total content (free + bound) of eight plant...

  15. A novel study of screening and confirmation of modafinil, adrafinil and their metabolite modafinilic acid under EI-GC-MS and ESI-LC-MS-MS ionization. (United States)

    Dubey, S; Ahi, S; Reddy, I M; Kaur, T; Beotra, A; Jain, S


    Adrafinil and modafinil have received wide publicity and have become controversial in the sporting world when several athletes were discovered allegedly using these drugs as doping agents. By acknowledging the facts, the World Anti-Doping Agency (WADA) banned these drugs in sports since 2004. The present study explores the possibility of differentiating adrafinil and modafinil and their major metabolites under electron impact ionization in gas chromatograph-mass spectrometer (GC-MSD) and electrospray ionization in liquid chromatograph-mass spectrometer (LC-MS/MS) by studying the fragmentation pattern of these drugs. Adrafinil, modafinil and their major metabolite, modafinilic acid were analyzed on EI-GC-MSD and ESI-LC-MS/MS using various individual parameters on both the instruments. The analytical technique and equipment used in the analysis were an Agilent 6890N GC with 5973 mass selective detector for the GC-MSD analysis and an Agilent 1100 HPLC with API-3200 Triple quadrupole mass spectrometer for the LC-MS/MS analysis. Validation of both methods was performed using six replicates at different concentrations. The results show that adrafinil, modafinil and their major metabolite modafinilic acid could be detected as a single artifact without differentiation under EI-GC-MSD analysis. However, all drugs could be detected and differentiated under ESI-LCMS/MS analysis without any artifaction. The GC-MSD analysis gives a single artifact for both the drugs without differentiation and thus can be used as a marker for screening purposes. Further, the Multiple Reaction Monitoring (MRM) method developed under LC-MS/MS is fit for the purpose for confirmation of suspicious samples in routine sports testing and in forensic and clinical analysis.

  16. LC/MS identification of 12 intracellular cytoskeletal and inflammatory proteins from monocytes adherent on surface-adsorbed fibronectin-derived peptides


    Zuckerman, Sean T.; Kao, Weiyuan John


    The extent and duration of the host response determines device efficacy yet the mechanism is poorly understood. U937 pro-monocytic cells were cultured on peptide-adsorbed tissue culture polystyrene to better understand surface-modulated intracellular events. Phosphotyrosine proteins were enriched by immunoprecipitation and analyzed by nanospray HPLC-coupled tandem mass spectrometry (LC/MS). Tyrosine phosphorylated proteins were chosen based on physiological significance and previous densitome...

  17. Development and validation of sensitive LC-MS/MS assays for quantification of HP-β-CD in human plasma and CSF. (United States)

    Jiang, Hui; Sidhu, Rohini; Fujiwara, Hideji; De Meulder, Marc; de Vries, Ronald; Gong, Yong; Kao, Mark; Porter, Forbes D; Yanjanin, Nicole M; Carillo-Carasco, Nuria; Xu, Xin; Ottinger, Elizabeth; Woolery, Myra; Ory, Daniel S; Jiang, Xuntian


    2-Hydroxypropyl-β-cyclodextrin (HP-β-CD), a widely used excipient for drug formulation, has emerged as an investigational new drug for the treatment of Niemann-Pick type C1 (NPC1) disease, a neurodegenerative cholesterol storage disorder. Development of a sensitive quantitative LC-MS/MS assay to monitor the pharmacokinetics (PKs) of HP-β-CD required for clinical trials has been challenging owing to the dispersity of the HP-β-CD. To support a phase 1 clinical trial for ICV delivery of HP-β-CD in NPC1 patients, novel methods for quantification of HP-β-CD in human plasma and cerebrospinal fluid (CSF) using LC-MS/MS were developed and validated: a 2D-LC-in-source fragmentation-MS/MS (2D-LC-IF-MS/MS) assay and a reversed phase ultra performance LC-MS/MS (RP-UPLC-MS/MS) assay. In both assays, protein precipitation and "dilute and shoot" procedures were used to process plasma and CSF, respectively. The assays were fully validated and in close agreement, and allowed determination of PK parameters for HP-β-CD. The LC-MS/MS methods are ∼100-fold more sensitive than the current HPLC assay, and were successfully employed to analyze HP-β-CD in human plasma and CSF samples to support the phase 1 clinical trial of HP-β-CD in NPC1 patients. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  18. LC-MS/MS method for hepcidin-25 measurement in human and mouse serum: clinical and research implications in iron disorders. (United States)

    Lefebvre, Thibaud; Dessendier, Nathalie; Houamel, Dounia; Ialy-Radio, Nathalie; Kannengiesser, Caroline; Manceau, Hana; Beaumont, Carole; Nicolas, Gael; Gouya, Laurent; Puy, Hervé; Karim, Zoubida


    The peptide hepcidin plays a central role in regulating dietary iron absorption and body iron distribution. This 25-amino acid hormone is produced and secreted predominantly by hepatocytes. Hepcidin has been suggested as a promising diagnostic marker for iron-related disorders. However, its accurate quantification for clinical use remains so far challenging. In this report we describe a highly specific and quantitative serum hepcidin method using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The analytical validation included the determination of the limit of detection, of quantification, repeatability, reproducibility and linearity. This assay was developed for human and mouse hepcidin. The human assay was performed on serum patients with unexplained microcytic anemia. We applied our LC-MS/MS method for quantifying hepcidin-1 in mouse in various conditions: inflammation, hemolytic anemia, Hamp-1, Hjv and Hfe KO mice. We show that the LC-MS/MS is suitable for accurate determination of hepcidin-25 in clinical samples, thereby representing a useful tool for the clinical diagnosis and follow-up of iron-related diseases. In mouse, a strong correlation between hepatic Hamp-1 mRNA expression and serum hepcidin-1 levels was found (r=0.88; p=0.0002) and the expected variations in mouse models of iron disorders were observed. Therefore, we propose this adaptive LC-MS/MS method as a suitable method for accurate determination of hepcidin-25 in clinical samples and as a major tool contributing to the clinical diagnosis, follow-up and management of iron-related disorders. It also opens new avenues to measure hepcidin in animal models without interspecies antigenic limitations.

  19. Comparative proteome analysis of egg yolk plasma proteins during storage. (United States)

    Gao, Dan; Qiu, Ning; Liu, Yaping; Ma, Meihu


    Physical changes such as chicken egg white thinning and egg yolk flattening occur during storage, implying a decline in egg quality. To reveal the deteriorative process related to chicken egg internal quality, a comparative proteomic method was used in this study to analyze the alterations in egg yolk plasma proteins at different storage times (0, 20 and 40 days) under an ambient temperature of 22 ± 2 °C. Using two-dimensional electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry, 33 protein spots representing 12 proteins were identified with significant (P albumin, vitellogenin fragments, IgY chains, ovalbumin, ovoinhibitor, α 2 -macroglobulin-like protein 1-like, hemopexin, transthyretin, apolipoprotein A-I and β 2 -glycoprotein I precursor. Accelerating degradation for most egg yolk plasma proteins was observed after prolonged storage (from day 20 to day 40). It is likely that the increased degradation of protease inhibitors such as ovoinhibitor and α 2 -macroglobulin-like protein 1-like during prolonged storage lead to an imbalance of protease and antiprotease in egg yolk, which may play a key role in the degradation of egg yolk proteins. These findings will provide an insight into the effects of storage on egg yolk protein changes and give a deeper understanding of the deteriorative process of chicken egg yolk. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  20. Identification of ractopamine glucuronides and determination of bioactive ractopamine residues and its metabolites in food animal urine by ELISA, LC-MS/MS and GC-MS. (United States)

    Jiang, Xiao-Fei; Zhu, Yue-Hui; Liu, Xiao-Yun


    Ractopamine glucuronides have been identified in cattle urine sampled by LC-MS/MS. An ELISA method, which was capable of specifically determining (1R, 3R)-ractopamine stereoisomer and its glucuronide metabolites, had more than 100% recovery with an acceptable coefficient of variation in the inter- and intra-assay variation tests for RR-ractopamine. The concentration levels of parent ractopamine and ractopamine glucuronide metabolites as the main components of total ractopamine in cattle and sheep urine showed similar depletion trends, in which the concentration curves increased and reached a climax during the feeding period, and then dropped quickly when entering the withdrawal period. Data from the three methods had very good pair-wise correlations. In the cattle urine samples, the correlation coefficient (R(2)) for parent ractopamine between the ELISA and the LC-MS/MS or GC-MS results were 0.93 or 0.92; R(2) values for parent ractopamine and total ractopamine data measured by LC-MS/MS and GC-MS were 0.9651 and 0.9677, respectively. All R(2) values for data gained from sheep urine samples were >0.95. The study indicated that the close levels of RR-ractopamine stereoisomer in cattle and sheep urine samples may imply the presence of a similar depletion pattern in other livestock, and thus would facilitate an accurate detection and management of ractopamine usage in food safety.

  1. Detailed LC-MS/MS analysis of ciguatoxins revealing distinct regional and species characteristics in fish and causative alga from the Pacific. (United States)

    Yogi, Kentaro; Oshiro, Naomasa; Inafuku, Yasuo; Hirama, Masahiro; Yasumoto, Takeshi


    Toxin profiles of representative ciguatera species caught at different locations of Japan were investigated in fish flesh by high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Identification and quantification of 16 toxins were facilitated by the use of 14 reference toxins prepared by either synthesis or isolation from natural sources and the previous LC-MS data thereof. Sodium adduct ions [M + Na](+) were used as parent and product ions. Distinct regional differences were unveiled: ciguatoxin-1B type toxins were found in snappers and groupers from Okinawa, ciguatoxin-3C type toxins were found in a spotted knifejaw, Oplegnathus punctatus, from Miyazaki located 730 km north of Okinawa, and both types of toxins were found in a red snapper, Lutjanus bohar, from Minamitorishima (Marcus) Island. Twelve toxins were identified in a dinoflagellate, Gambierdiscus toxicus, collected as the primary toxin source in French Polynesia. Occurrence of M-seco-toxins in fish and oxidized toxins in the dinoflagellate was confirmed for the first time. The present LC-MS/MS method is rapid, specific, and accurate. It not only outperforms the currently employed mouse bioassays but also enables the study of the toxin dynamics during the food chain transmission.

  2. Improvement of Nicotinic Acid and Nicotinamide Analysis in Meats and Meat Products by HPLC and LC-MS/MS with Solid-Phase Extraction. (United States)

    Hiki, Asako; Yamajima, Yukiko; Uematsu, Yoko


    A method for nicotinic acid (NA) and nicotinamide (NAA) analysis in meats was developed. NA and NAA were extracted from meats or meat products with metaphosphate aqueous solution. The extract was cleaned up with an Oasis MCX cartridge. The cartridge was washed with 2% acetic acid (v/v) and acetic acid-methanol solution. NA and NAA were eluted with ammonia-methanol solution. NA and NAA in the eluate were chromatographed on a Scherzo SM-C18 (3.0×150 mm, 3.0 μm) column with 20 mmol/L ammonium acetate containing 0.1% acetic acid-acetonitrile (97 : 3) as a mobile phase and were monitored at 261 nm. Quantification was performed by LC and LC-MS/MS. Calibration curves showed high linearity (correlation coefficient>0.998) between 1-25 μg/mL for LC and LC-MS/MS. Recoveries were 84-108% (CV≦5.8%) by HPLC and 79-105% (CV≦9.0%) by LC-MS/MS. The limit of quantitation for NA was 0.005-0.01 g/kg and that for NAA was 0.01-0.02 g/kg.

  3. Identification of the Chemical Constituents in Simiao Wan and Rat Plasma after Oral Administration by GC-MS and LC-MS

    Directory of Open Access Journals (Sweden)

    Yunshuang Fan


    Full Text Available Simiao Wan (SMW, an important multiherbal formula used in traditional Chinese medicine, is extensively used to treat rheumatoid arthritis. However, the knowledge of the bioactive components of SMW remains unclear. Thus, gas chromatography–mass spectrometry (GC-MS and liquid chromatography–mass spectrometry (LC-MS were used to analyze the chemical constituents of volatile and nonvolatile extracts of SMW, as well as its absorbed components in rat plasma after oral SMW administration. Identification of several compounds was enabled by comparison of retention times, MS spectra, and MS/MS spectral data with the standard substance and reference materials reported in the literature. In the volatile extracts, GC-MS identified 26 compounds in vitro, three of which observed in blood by GC-MS. In the nonvolatile extracts, LC-MS identified 49 compounds in SMW; 18 compounds containing 7 prototype compounds, 5 metabolites, and 6 unknown compounds were absorbed by blood. The proposed GC-MS and LC-MS method was appropriate not only for the rapid screening and identification of multiple components of an SMW extract but also for screening its bioactive constituents in vivo. The proposed method could be a promising tool for the quality control of other Chinese herbal medicines.

  4. Isolation of α-Amylase Inhibitors from Kadsura longipedunculata Using a High-Speed Counter-Current Chromatography Target Guided by Centrifugal Ultrafiltration with LC-MS

    Directory of Open Access Journals (Sweden)

    Yin Cen


    Full Text Available In this study, a high-speed counter-current chromatography (HSCCC separation method target guided by centrifugal ultrafiltration with high-performance liquid chromatography-mass spectrometry (CU-LC-MS was proposed. This method was used to analyze α-amylase inhibitors from Kadsura longipedunculata extract. According to previous screening with CU-LC-MS, two screened potential α-amylase inhibitors was successfully isolated from Kadsura longipedunculata extract using HSCCC under the optimized experimental conditions. The isolated two target compounds (with purities of 92.3% and 94.6% were, respectively, identified as quercetin-3-O-rhamnoside (1 and protocatechuic acid (2 based on the MS, UV, and 1H-NMR spectrometry data. To verify the inhibition of screened compounds, the inhibitory activities of quercetin-3-O-rhamnoside (1 and protocatechuic acid (2 on α-amylase were tested, and it demonstrated that the experimental IC50 values of quercetin-3-O-rhamnoside (1 and protocatechuic acid (2 were 28.8 and 12.5 μmol/L. These results proved that the hyphenated technique using CU-LC-MS and HSCCC was a rapid, competent, and reproductive method to screen and separate potential active compounds, like enzyme inhibitors from the extract of herbal medicines.

  5. Isolation of α-Amylase Inhibitors from Kadsura longipedunculata Using a High-Speed Counter-Current Chromatography Target Guided by Centrifugal Ultrafiltration with LC-MS. (United States)

    Cen, Yin; Xiao, Aiping; Chen, Xiaoqing; Liu, Liangliang


    In this study, a high-speed counter-current chromatography (HSCCC) separation method target guided by centrifugal ultrafiltration with high-performance liquid chromatography-mass spectrometry (CU-LC-MS) was proposed. This method was used to analyze α-amylase inhibitors from Kadsura longipedunculata extract. According to previous screening with CU-LC-MS, two screened potential α-amylase inhibitors was successfully isolated from Kadsura longipedunculata extract using HSCCC under the optimized experimental conditions. The isolated two target compounds (with purities of 92.3% and 94.6%) were, respectively, identified as quercetin-3-O-rhamnoside (1) and protocatechuic acid (2) based on the MS, UV, and ¹H-NMR spectrometry data. To verify the inhibition of screened compounds, the inhibitory activities of quercetin-3-O-rhamnoside (1) and protocatechuic acid (2) on α-amylase were tested, and it demonstrated that the experimental IC50 values of quercetin-3-O-rhamnoside (1) and protocatechuic acid (2) were 28.8 and 12.5 μmol/L. These results proved that the hyphenated technique using CU-LC-MS and HSCCC was a rapid, competent, and reproductive method to screen and separate potential active compounds, like enzyme inhibitors from the extract of herbal medicines.

  6. Combination of pentafluorophenylhydrazine derivatization and isotope dilution LC-MS/MS techniques for the quantification of apurinic/apyrimidinic sites in cellular DNA. (United States)

    Li, Jie; Leung, Elvis M K; Choi, Martin M F; Chan, Wan


    Apurinic/apyrimidinic (AP) sites are common DNA lesions arising from spontaneous hydrolysis of the N-glycosidic bond and base-excision repair mechanisms of the modified bases. Due to the strong association of AP site formation with physically/chemically induced DNA damage, quantifying AP sites provides important information for risk assessment of exposure to genotoxins and oxidative stress. However, rigorous quantification of AP sites in DNA has been hampered by technical problems relating to the sensitivity and selectivity of existing analytical methods. We have developed a new isotope dilution liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) method for the rigorous quantification of AP sites in genomic DNA. The method entails enzymatic digestion of AP site-containing DNA by endo- and exonucleases, derivatization with pentafluorophenylhydrazine (PFPH), addition of an isotopically labeled PFPH derivative as internal standard, and quantification by LC-MS/MS. The combination of PFPH derivatization with LC-MS/MS analysis on a triple quadrupole mass spectrometer allows for sensitive and selective quantification of AP sites in DNA at a detection limit of 6.5 fmol, corresponding to 4 AP sites/10(9) nt in 5 μg of DNA, which is at least ten times more sensitive than existing analytical methods. The protocol was validated by AP site-containing oligonucleotides and applied in quantifying methyl methanesulfonate-induced formation of AP sites in cellular DNA.

  7. Identification and quantification of glucosinolate and flavonol compounds in rocket salad (Eruca sativa, Eruca vesicaria and Diplotaxis tenuifolia) by LC-MS: highlighting the potential for improving nutritional value of rocket crops. (United States)

    Bell, Luke; Oruna-Concha, Maria Jose; Wagstaff, Carol


    Liquid chromatography mass spectrometry (LC-MS) was used to obtain glucosinolate and flavonol content for 35 rocket accessions and commercial varieties. 13 glucosinolates and 11 flavonol compounds were identified. Semi-quantitative methods were used to estimate concentrations of both groups of compounds. Minor glucosinolate composition was found to be different between accessions; concentrations varied significantly. Flavonols showed differentiation between genera, with Diplotaxis accumulating quercetin glucosides and Eruca accumulating kaempferol glucosides. Several compounds were detected in each genus that have only previously been reported in the other. We highlight how knowledge of phytochemical content and concentration can be used to breed new, nutritionally superior varieties. We also demonstrate the effects of controlled environment conditions on the accumulations of glucosinolates and flavonols and explore the reasons for differences with previous studies. We stress the importance of consistent experimental design between research groups to effectively compare and contrast results. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. Comparative Proteomic Analysis of the Mitochondria-associated ER Membrane (MAM) in a Long-term Type 2 Diabetic Rodent Model. (United States)

    Ma, Jacey Hongjie; Shen, Shichen; Wang, Joshua J; He, Zhanwen; Poon, Amanda; Li, Jun; Qu, Jun; Zhang, Sarah X


    The mitochondria-associated ER membrane (MAM) plays a critical role in cellular energetics and calcium homeostasis; however, how MAM is affected under diabetic condition remains elusive. This study presented a comprehensive proteome profiling of isolated brain MAM from long-term type 2 diabetic mice vs. non-diabetic controls. MAM protein was extracted efficiently by a surfactant-aided precipitation/on-pellet digestion (SOD) method, and MAM proteome was quantified by an ion-current-based MS1 method combined with nanoLC-MS/MS. A total of 1,313 non-redundant proteins of MAM were identified, among which 144 proteins were found significantly altered by diabetes. In-depth IPA analysis identified multiple disease-relevant signaling pathways associated with the MAM proteome changes in diabetes, most significantly the unfolded protein response (UPR), p53, hypoxia-related transcription factors, and methyl CpG binding protein 2. Using immunofluorescence labeling we confirmed the activation of three UPR branches and increased ERp29 and calreticulin in diabetic retinas. Moreover, we found GRP75, a key MAM tethering protein, was drastically reduced by long-term diabetes. In vitro, acute high glucose treatment reduces ER-mitochondrial contact in retinal endothelial cells. This study provides first insight into the significant alterations in MAM proteome associated with activation of the UPR in diabetes, which may serve as novel benchmarks for the future studies of diabetic complications.

  9. Use of isotope-labeled aflatoxins for LC-MS/MS stable isotope dilution analysis of foods. (United States)

    Cervino, Christian; Asam, Stefan; Knopp, Dietmar; Rychlik, Michael; Niessner, Reinhard


    Aflatoxins are a group of very carcinogenic mycotoxins that can be found on a wide range of food commodities including nuts, cereals, and spices. In this study, the first LC-MS/MS stable isotope dilution assay (SIDA) for the determination of aflatoxins in foods was developed. The development of this method was enabled by easily accessible isotope-labeled (deuterated) aflatoxins B2 and G2, which were synthesized by catalytic deuteration of aflatoxin B1 and G1, purified, and well-characterized by NMR and MS. All four aflatoxins of interest (B1, B2, G1, and G2) were quantified in food samples by using these two labeled internal standards. The response factors (RF) of the linear calibrations were revealed to be matrix independent for labeled aflatoxin B2/aflatoxin B2 and labeled aflatoxin G2/aflatoxin G2. For labeled aflatoxin B 2/aflatoxin B 1 and labeled aflatoxin B2/aflatoxin G1 matrix-matched calibration was performed for the model matrices almonds and wheat flour, showing significant differences of the RFs. Limits of detection (LOD) were determined by applying a statistical approach in the presence of the two model matrices, yielding 0.31 microg/kg (aflatoxin B1), 0.09 microg/kg (aflatoxin B2), 0.38 microg/kg (aflatoxin G1), and 0.32 microg/kg (aflatoxin G2) for almonds (similar LODs were obtained for wheat flour). Recovery rates were between 90 and 105% for all analytes. Coefficients of variation (CV) of 12% (aflatoxin B1), 3.6% (aflatoxin B2), 14% (aflatoxin G1), and 4.8% (aflatoxin G2) were obtained from interassay studies. For further validation, a NIST standard reference food sample was analyzed for aflatoxins B1 and B2. The method was successfully applied to determine trace levels of aflatoxins in diverse food matrices such as peanuts, nuts, grains, and spices. Aflatoxin contents in these samples ranged from about 0.5 to 6 microg/kg.

  10. Simultaneous determination of riboflavin and pyridoxine by UHPLC/LC-MS in UK commercial infant meal food products. (United States)

    Zand, Nazanin; Chowdhry, Babur Z; Pullen, Frank S; Snowden, Martin J; Tetteh, John


    An assay for the simultaneous quantitative determination of riboflavin and pyridoxine in eight different complementary infant meal products has been developed in order to (1) estimate the daily intake of these vitamins from commercial infant food consumption, and (2) ascertain their nutritional suitability relative to dietary guidelines for the 6-9 months age group. The method involves mild hydrolysis of the foods, an extraction of the supernatant by centrifugation followed by quantitative determination using ultra-high performance liquid chromatography. Separation of the two water soluble vitamins is achieved within one minute and the resultant sample is also LC-MS compatible. Despite wide individual differences between brands (p=6.5e-12), no significant differences were observed in the level of pyridoxine between the meat and vegetable-based varieties (p=0.7) per 100g of commercial infant food. Riboflavin was not detected in any of the samples where the detection limit was below 0.07 μg/mL. In terms of the Reference Nutrient Intake (RNI) of pyridoxine for 6-9 months old infants, the complementary infant meal products analysed herein provided less than 15% of the RNI values with mean (SD) values of 12.87 (± 4.46)% and 13.88 (± 4.97)% for the meat- and vegetable-based recipes, respectively. The estimated total daily intake of riboflavin and pyridoxine from the consumption of commercial complementary food was found to be satisfactory and in accordance with the Dietary Reference Values (DRVs). The intake of both riboflavin and pyridoxine was estimated to be mainly derived from the consumption of formula milk which could be a cause of concern if the quality of an infant's milk diet is compromised by an inadequate or lack of supplemented milk intake. The results of this study suggest that the selected commercial complementary infant foods in the UK market may not contain the minimum levels of riboflavin and pyridoxine required for the labelling declaration of the

  11. Measurement of atropine and scopolamine in hair by LC-MS/MS after Datura stramonium chronic exposure. (United States)

    Ricard, Florian; Abe, Emuri; Duverneuil-Mayer, Charlotte; Charlier, Philippe; de la Grandmaison, Geoffroy; Alvarez, Jean Claude


    Datura stramonium is an herbaceous annual plant. All parts of the plant contain tropane alkaloids such as atropine and scopolamine. We report the case of a 22-year-old man admitted to a general hospital for visual and aural hallucinations. One week after his admission, as the hallucinations remained, the patient was transferred to a psychiatric hospital. Neither blood nor urine was conserved during his hospitalization, so a hair analysis was requested in order to identify a possible consumption of a Datura seed infusion. After decontamination and washing, hair strands were segmented into four pieces and grinded into a fine and homogeneous powder. We then incubated 20 mg for 10 min in 1 mL of phosphate buffer at pH 5.0 in the presence of 100 ng of ketamine-d4, used as internal standard (IS). Liquid-liquid extraction was performed with 4 mL of a mixture of hexane/ethyl acetate (1/1, v/v). The residue was reconstituted in 80 μL of mobile phase. A further 10 μL were injected into an 1.9 μm Hypersil GOLD PFP column (100 mm×2.1 mm) eluted with a gradient of acetonitrile and 2 mmol/L 0.1% formate buffer at a flow rate of 300 μL/min. Compounds were detected by a LCQ TSQ Vantage XP triple-quadripole mass spectrometer equipped with an electrospray ionization (ESI) source set in positive mode. SRM transitions m/z 290.2→124.1, m/z 304.2→138.1, and m/z 242.1→129.1 were optimized for atropine, scopolamine and IS, respectively. The assay was accurate and precise over the range of 1.0 (lower limit of quantification) to 1000.0 pg/mg (upper limit of quantification) in hair. Both atropine (from 8.4 to 15.0 pg/mg) and scopolamine (1.0-1.3 pg/mg) were identified in the four segment of the hair showing a regular consumption of Datura admitted by the patient himself. We report here the first description of atropine with scopolamine in a Caucasian dark man's hair after D. stramonium chronic exposure, using a validated LC-MS/MS method. Copyright © 2012 Elsevier Ireland Ltd. All

  12. Enantiospecific determination of arotinolol in rat plasma by LC-MS/MS: application to a stereoselective pharmacokinetic study. (United States)

    Qian, Zheyuan; Xu, Yanhai; Zheng, Leyi; Zhang, Jingbo; Hong, Zhanying; Shen, Xiaohang


    A highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and fully validated for quantification of arotinolol enantiomers in rat plasma using haloperidol as the internal standard. After solid phase extraction of 50.0 μL rat plasma in 96 well plate, a baseline resolution of arotinolol enantiomers was achieved on a CHIRALPAK AD-H column using the mobile phase of n-hexane and ethanol in 0.02% diethylamine (20:80, v/v) at a flow rate of 0.550 mL/min within 11.0 min. Acquisition of mass spectrometric data was performed on a triple-quadrupole mass spectrometer in multiple-reaction-monitoring (MRM) mode with an ESI source using the transition m/z 372.1 → 316.1 for (±)-arotinolol and m/z 376.1 → 165.1 for haloperidol. The calibration curves of both enantiomers were linear over the range of 1.00-200.0 ng/mL (r(2)>0.992) and the lower limit of quantification was 1.00 ng/mL. Intra- and inter-day precision ranged from 5.6% to 8.9% for R-(-)-arotinolol and 4.6-7.4% for S-(+)-arotinolol. Accuracy varied from 0.0% to 7.0% for R-(-)-arotinolol and 5.0-10.0% for S-(+)-arotinolol. For R-(-)-arotinolol, the recovery ranged from 87.2% to 99.2% and the matrix factor was 1.03-1.09; for S-(+)-arotinolol, the recovery ranged from 88.0% to 92.4% and the matrix factor was 0.84-0.95, both were not concentration dependent. The method was demonstrated with acceptable accuracy, precision and specificity for the determination of arotinolol enantiomers and has been successfully applied to a stereoselective pharmacokinetic study. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Comparative Proteomics of Mouse Tears and Saliva: Evidence from Large Protein Families for Functional Adaptation

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    Robert C. Karn


    Full Text Available We produced a tear proteome of the genome mouse, C57BL/6, that contained 139 different protein identifications: 110 from a two-dimensional (2D gel with subsequent trypsin digestion, 19 from a one-dimensional (1D gel with subsequent trypsin digestion and ten from a 1D gel with subsequent Asp-N digestion. We compared this tear proteome with a C57BL/6 mouse saliva proteome produced previously. Sixteen of the 139 tear proteins are shared between the two proteomes, including six proteins that combat microbial growth. Among the 123 other tear proteins, were members of four large protein families that have no counterparts in humans: Androgen-binding proteins (ABPs with different members expressed in the two proteomes, Exocrine secreted peptides (ESPs expressed exclusively in the tear proteome, major urinary proteins (MUPs expressed in one or both proteomes and the mouse-specific Kallikreins (subfamily b KLKs expressed exclusively in the saliva proteome. All four families have members with suggested roles in mouse communication, which may influence some aspect of reproductive behavior. We discuss this in the context of functional adaptation involving tear and saliva proteins in the secretions of mouse lacrimal and salivary glands, respectively.

  14. Characterization of the gila monster (Heloderma suspectum suspectum) venom proteome


    Sanggaard, Kristian W.; Dyrlund, Thomas F.; Thomsen, Line R.; Nielsen, Tania A.; Brøndum, Lars; Wang, Tobias; Thøgersen, Ida B.; Enghild, Jan J.


    The data presented here is related to the research article entitled ?Characterization of the gila monster (Heloderma suspectum suspectum) venom proteome? by Sanggaard et al. in Journal of Proteomics [1]. The gila monster venom was collected, analyzed by 2D-gel electrophoresis and after Coomassie-Brilliant Blue staining the major spots were excised, subjected to in-gel trypsin digestion, and analyzed by LC?MS/MS. Subsequently, the venom proteins were identified based on de novo sequencing and ...

  15. An attempt to understand kidney's protein handling function by comparing plasma and urine proteomes.

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    Lulu Jia

    Full Text Available BACKGROUND: With the help of proteomics technology, the human plasma and urine proteomes, which closely represent the protein compositions of the input and output of the kidney, respectively, have been profiled in much greater detail by different research teams. Many datasets have been accumulated to form "reference profiles" of the plasma and urine proteomes. Comparing these two proteomes may help us understand the protein handling aspect of kidney function in a way, however, which has been unavailable until the recent advances in proteomics technology. METHODOLOGY/PRINCIPAL FINDINGS: After removing secreted proteins downstream of the kidney, 2611 proteins in plasma and 1522 in urine were identified with high confidence and compared based on available proteomic data to generate three subproteomes, the plasma-only subproteome, the plasma-and-urine subproteome, and the urine-only subproteome, and they correspond to three groups of proteins that are handled in three different ways by the kidney. The available experimental molecular weights of the proteins in the three subproteomes were collected and analyzed. Since the functions of the overrepresented proteins in the plasma-and-urine subproteome are probably the major functions that can be routinely regulated by excretion from the kidney in physiological conditions, Gene Ontology term enrichment in the plasma-and-urine subproteome versus the whole plasma proteome was analyzed. Protease activity, calcium and growth factor binding proteins, and coagulation and immune response-related proteins were found to be enriched. CONCLUSION/SIGNIFICANCE: The comparison method described in this paper provides an illustration of a new approach for studying organ functions with a proteomics methodology. Because of its distinctive input (plasma and output (urine, it is reasonable to predict that the kidney will be the first organ whose functions are further elucidated by proteomic methods in the near future. It

  16. Development of a fast isocratic LC-MS/MS method for the high-throughput analysis of pyrrolizidine alkaloids in Australian honey. (United States)

    Griffin, Caroline T; Mitrovic, Simon M; Danaher, Martin; Furey, Ambrose


    Honey samples originating from Australia were purchased and analysed for targeted pyrrolizidine alkaloids (PAs) using a new and rapid isocratic LC-MS/MS method. This isocratic method was developed from, and is comparable with, a gradient elution method and resulted in no loss of sensitivity or reduction in chromatographic peak shape. Isocratic elution allows for significantly shorter run times (6 min), eliminates the requirement for column equilibration periods and, thus, has the advantage of facilitating a high-throughput analysis which is particularly important for regulatory testing laboratories. In excess of two hundred injections are possible, with this new isocratic methodology, within a 24-h period which is more than 50% improvement on all previously published methodologies. Good linear calibrations were obtained for all 10 PAs and four PA N-oxides (PANOs) in spiked honey samples (3.57-357.14 µg l(-1); R(2) ≥ 0.9987). Acceptable inter-day repeatability was achieved for the target analytes in honey with % RSD values (n = 4) less than 7.4%. Limits of detection (LOD) and limits of quantitation (LOQ) were achieved with spiked PAs and PANOs samples; giving an average LOD of 1.6 µg kg(-1) and LOQ of 5.4 µg kg(-1). This method was successfully applied to Australian and New Zealand honey samples sourced from supermarkets in Australia. Analysis showed that 41 of the 59 honey samples were contaminated by PAs with the mean total sum of PAs being 153 µg kg(-1). Echimidine and lycopsamine were predominant and found in 76% and 88%, respectively, of the positive samples. The average daily exposure, based on the results presented in this study, were 0.051 µg kg(-1) bw day(-1) for adults and 0.204 µg kg(-1) bw day(-1) for children. These results are a cause for concern when compared with the proposed European Food Safety Authority (EFSA), Committee on Toxicity (COT) and Bundesinstitut für Risikobewertung (BfR - Federal Institute of Risk Assessment Germany) maximum

  17. LC-MS/MS as an alternative for SDS-PAGE in blue native analysis of protein complexes.

    NARCIS (Netherlands)

    Wessels, H.C.T.; Vogel, R.O.; Heuvel, L.P.W.J. van den; Smeitink, J.A.M.; Rodenburg, R.J.T.; Nijtmans, L.G.J.; Farhoud, M.H.


    Two-dimensional blue native/SDS-PAGE is widely applied to investigate native protein-protein interactions, particularly those within membrane multi-protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we

  18. Comprehensive Proteomic Analysis of Human Milk-derived Extracellular Vesicles Unveils a Novel Functional Proteome Distinct from Other Milk Components* (United States)

    van Herwijnen, Martijn J.C.; Zonneveld, Marijke I.; Goerdayal, Soenita; Nolte – 't Hoen, Esther N.M.; Garssen, Johan; Stahl, Bernd; Maarten Altelaar, A.F.; Redegeld, Frank A.; Wauben, Marca H.M.


    Breast milk contains several macromolecular components with distinctive functions, whereby milk fat globules and casein micelles mainly provide nutrition to the newborn, and whey contains molecules that can stimulate the newborn's developing immune system and gastrointestinal tract. Although extracellular vesicles (EV) have been identified in breast milk, their physiological function and composition has not been addressed in detail. EV are submicron sized vehicles released by cells for intercellular communication via selectively incorporated lipids, nucleic acids, and proteins. Because of the difficulty in separating EV from other milk components, an in-depth analysis of the proteome of human milk-derived EV is lacking. In this study, an extensive LC-MS/MS proteomic analysis was performed of EV that had been purified from breast milk of seven individual donors using a recently established, optimized density-gradient-based EV isolation protocol. A total of 1963 proteins were identified in milk-derived EV, including EV-associated proteins like CD9, Annexin A5, and Flotillin-1, with a remarkable overlap between the different donors. Interestingly, 198 of the identified proteins are not present in the human EV database Vesiclepedia, indicating that milk-derived EV harbor proteins not yet identified in EV of different origin. Similarly, the proteome of milk-derived EV was compared with that of other milk components. For this, data from 38 published milk proteomic studies were combined in order to construct the total milk proteome, which consists of 2698 unique proteins. Remarkably, 633 proteins identified in milk-derived EV have not yet been identified in human milk to date. Interestingly, these novel proteins include proteins involved in regulation of cell growth and controlling inflammatory signaling pathways, suggesting that milk-derived EVs could support the newborn's developing gastrointestinal tract and immune system. Overall, this study provides an expansion of

  19. HPTLC-profiling of eleutherosides, mechanism of antioxidative action of eleutheroside E1, the PAMPA test with LC/MS detection and the structure–activity relationship

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    Daniel Załuski


    Full Text Available Human body is constantly generating free radicals, which causes oxidative stress. Despite naturally occurring antioxidant systems in human body, free radicals cause lipid, proteins and DNA oxidation. New antioxidants are still urgent as well as their mechanisms of action should be explained. In this study, we investigated the mechanism by which eleutherosides B, E and E1 may act as antioxidants, identified eleutherosides in Eleutherococcus lasiogyne and Eleutherococcus giraldii, and explained in vitro the absorption of eleutheroside E1 based on passive transport. The DPPH∗ and DB-HPTLC tests were used to assess the antioxidant activity. Of the three eleutherosides, only eleutheroside E1 exhibited a strong anti-DPPH∗ activity (EC50 37.03 μg/mL; 63 mMol compared to the raw extracts (EC50 170 and 180 μg/mL for E. lasiogyne and E. giraldii. This activity was also confirmed by the DB-HPTLC autography technique. According to Załuski’s hypothesis, the antioxidant mechanism of eleutheroside E1 is based on the complexation of DPPH∗ molecule with its aryl radical. During this reaction, the aryl radical of eleutheroside E1 (E1∗ and DPPHH are created. Next, the aryl radical (E1∗ is complexed with another DPPH∗ molecule. Additionally, the aryl radical can be stabilized by the presence of the methoxy groups in the aromatic ring, which increases its antioxidative action. The HPTLC-identification of extracts showed the presence of eleutherosides B, E and E1 in both species. The PAMPA test coupled with LC/MS detection showed a low permeability of eleutheroside E1 across artificial membrane. Because eleutherosides belong to the polyphenols, the TPC and TFC were quantified. The TPC and TFC varied from 51.4 to 49.3 mg/g dry extract for TPC, and from 5.73 to 4.91 mg/g dry extract for TFC, for E. giraldii and E. lasiogyne, respectively. In conclusion, eleutheroside E1 in its pure form could be a chemopreventive ingredient of new pharmacological

  20. Development of in-house ELISA for detection of chloramphenicol in bovine milk with subsequent confirmatory analysis by LC-MS/MS. (United States)

    Chughtai, Muhammad I; Maqbool, Uzma; Iqbal, Mazhar; Shah, Muhammad S; Fodey, Terence


    This study was undertaken to develop and validate direct competitive ELISA for the determination of chloramphenicol residues in bovine milk. Antisera and an enzyme-tracer for chloramphenicol were prepared and used to develop an ELISA with inhibition concentrations, IC 20 and IC 50 , of 0.09 and 0.44 ng mL -1 , respectively. Milk samples were spiked with standards equivalent to 0, 0.2, 0.3, 0.5, 1.0 & 1.5 ng mL -1 and extracted in methanol. The mean recoveries were found to be 73-100% with coefficient of variance 7-11%. The decision limit (CCα) and detection capability (CCβ) were calculated as 0.10 and 0.12 ng mL -1 , respectively. The results were found comparable with the commercial ELISA, having recoveries of 87 to 100%, CCα 0.09 ng mL -1 and CCβ 0.12 ng mL -1 . As per Commission Decision 2002/657/EC, in-house ELISA was further validated by using LC-MS/MS. Mass spectral acquisition was done by using electrospray ionization in the negative ion mode applying single reaction monitoring of the diagnostic transition reaction for CAP (m/z 152, 194 and 257). The calibration curve showed good linearity in concentrations from 0.025 to 1.6 ng mL -1 with correction coefficient 0.9902. The mean recoveries were found to be 88 to 100%. The CCα was calculated as 0.057 ng mL -1 and CCβ 0.10 ng mL -1 . Since CCα and CCβ are less than half of the MRPL (0.15 ng mL -1 ), the test was found suitable for screening and quantification of CAP residues in bovine milk samples. Results of surveillance studies indicated that out of 31 analyzed milk samples, 12.9% samples were found with CAP residues but only 3.2% samples were declared positive with maximum concentration 0.31 ng mL -1 , slightly above the MRPL.

  1. Quantitative chiral and achiral determination of ketamine and its metabolites by LC-MS/MS in human serum, urine and fecal samples. (United States)

    Hasan, Mahmoud; Hofstetter, Robert; Fassauer, Georg M; Link, Andreas; Siegmund, Werner; Oswald, Stefan


    Ketamine (KET) is a widely used anesthetic drug which is metabolized by CYP450 enzymes to norketamine (n-KET), dehydronorketamine (DHNK), hydroxynorketamine (HNK) and hydroxyketamine (HK). Ketamine is a chiral compound and S-ketamine is known to be the more potent enantiomer. Here, we present the development and validation of three LC-MS/MS assays; the first for the quantification of racemic KET, n-KET and DHNK in human serum, urine and feces; the second for the separation and quantification of the S- and R-enantiomers of KET, n-KET and DHNK, and the third for separation and quantification of 2S,6S-hydroxynorketamine (2S,6S-HNK) and 2R,6R-hydroxynorketamine (2R,6R-HNK) in serum and urine with the ability to separate and detect 10 additional hydroxylated norketamine metabolites of racemic ketamine. Sample preparation was done by liquid-liquid extraction using methyl tert-butyl ether. For achiral determination of KET and its metabolites, an isocratic elution with ammonium acetate (pH 3.8; 5mM) and acetonitrile on a C18 column was performed. For the separation of S- and R-enantiomers of KET, n-KET and DHNK, a gradient elution was applied using a mobile phase of ammonium acetate (pH 7.5; 10mM) and isopropanol on the CHIRAL-AGP ® column. The enantioselective separation of the HNK metabolites was done on the chiral column Lux ® -Amylose-2 with a gradient method using ammonium acetate (pH 9; 5mM) and a mixture of isopropanol and acetonitrile (4:1). The mass spectrometric detection monitored for each analyte 2-3 mass/charge transitions. D4-ketamine and D4-n-KET were used as internal standards. The assays were successfully validated according to current bioanalytical guidelines and applied to a pilot study in one healthy volunteer. Compared to previously published methods, our assays have superior analytical features such as a lower amount of required matrix, faster sample preparation, shorter analytical run time and higher sensitivity (LLOQ up to 0.1ng/ml). Moreover, our

  2. Incidence of pharmaceuticals in soils, sediments and waters of Pego-Oliva Marsh by LC-MS/MS. (United States)

    Vazquez-Roig, P.; Andreu, V.; Blasco, C.; Picó, Y.


    The presence of pharmaceutical residues in the environmental compartments is a growing problem that could have unexpected consequences. In recent years, the number of pharmaceuticals detected in the environment had increased spectacularly, reaching a broad number of the most consumed drugs and including virtually all the existing therapeutic classes. These compounds come mainly from human excretions, waste effluents of manufacturing processes and animal farms. In Spain, obsolete sewage treatment plants, and even the absence of those, are the main problem to be solved. Some pharmaceuticals have shown toxicity to bacteria, algae and invertebrates. Besides that reproductive problems in fishes have been observed in "in vitro" studies. By the other hand, synergistic effects of exposure to mixtures of drugs or toxic effects due to accumulation would be expected. A method developed in our laboratory was utilized to monitor the occurrence of 16 relevant pharmaceuticals in the Pego-Oliva Marsh Natural Reserve (Valencian Community, Spain). A total 46 samples of soils (at two different depths), 15 sediments and 34 waters were collected in June 2009. Solid samples were concentrated by pressurized liquid extraction (ASE® 200) using water at 90°C as extracting solvent and three cycles of extraction of 7 minutes. The aqueous extract obtained was passed through two cartridges connected in series: to an Isolute® SAX cartridge (strong anion exchange) on the top and an Oasis® HLB cartridge below. Extraction was carried out with 6mL of methanol. Quantification was performed by a Quattro Micro LC-MS/MS with an ESI interface working in both positive and negative mode. Two transitions were utilized for each compound to obtain an unequivocal confirmation, with the exception of ibuprofen which only gave one transition with adequate sensitivity. All water samples appeared contaminated with at least with two compounds. Ibuprofen and codeine were the compounds more frequently detected in

  3. Technical advances in proteomics: new developments in data-independent acquisition [version 1; referees: 3 approved

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    Alex Hu


    Full Text Available The ultimate aim of proteomics is to fully identify and quantify the entire complement of proteins and post-translational modifications in biological samples of interest. For the last 15 years, liquid chromatography-tandem mass spectrometry (LC-MS/MS in data-dependent acquisition (DDA mode has been the standard for proteomics when sampling breadth and discovery were the main objectives; multiple reaction monitoring (MRM LC-MS/MS has been the standard for targeted proteomics when precise quantification, reproducibility, and validation were the main objectives. Recently, improvements in mass spectrometer design and bioinformatics algorithms have resulted in the rediscovery and development of another sampling method: data-independent acquisition (DIA. DIA comprehensively and repeatedly samples every peptide in a protein digest, producing a complex set of mass spectra that is difficult to interpret without external spectral libraries. Currently, DIA approaches the identification breadth of DDA while achieving the reproducible quantification characteristic of MRM or its newest version, parallel reaction monitoring (PRM. In comparative de novo identification and quantification studies in human cell lysates, DIA identified up to 89% of the proteins detected in a comparable DDA experiment while providing reproducible quantification of over 85% of them. DIA analysis aided by spectral libraries derived from prior DIA experiments or auxiliary DDA data produces identification and quantification as reproducible and precise as that achieved by MRM/PRM, except on low‑abundance peptides that are obscured by stronger signals. DIA is still a work in progress toward the goal of sensitive, reproducible, and precise quantification without external spectral libraries. New software tools applied to DIA analysis have to deal with deconvolution of complex spectra as well as proper filtering of false positives and false negatives. However, the future outlook is

  4. ProteomicsDB. (United States)

    Schmidt, Tobias; Samaras, Patroklos; Frejno, Martin; Gessulat, Siegfried; Barnert, Maximilian; Kienegger, Harald; Krcmar, Helmut; Schlegl, Judith; Ehrlich, Hans-Christian; Aiche, Stephan; Kuster, Bernhard; Wilhelm, Mathias


    ProteomicsDB ( is a protein-centric in-memory database for the exploration of large collections of quantitative mass spectrometry-based proteomics data. ProteomicsDB was first released in 2014 to enable the interactive exploration of the first draft of the human proteome. To date, it contains quantitative data from 78 projects totalling over 19k LC-MS/MS experiments. A standardized analysis pipeline enables comparisons between multiple datasets to facilitate the exploration of protein expression across hundreds of tissues, body fluids and cell lines. We recently extended the data model to enable the storage and integrated visualization of other quantitative omics data. This includes transcriptomics data from e.g. NCBI GEO, protein-protein interaction information from STRING, functional annotations from KEGG, drug-sensitivity/selectivity data from several public sources and reference mass spectra from the ProteomeTools project. The extended functionality transforms ProteomicsDB into a multi-purpose resource connecting quantification and meta-data for each protein. The rich user interface helps researchers to navigate all data sources in either a protein-centric or multi-protein-centric manner. Several options are available to download data manually, while our application programming interface enables accessing quantitative data systematically. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.