A systematic evaluation of normalization methods in quantitative label-free proteomics.

Science.gov (United States)

Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

2018-01-01

To date, mass spectrometry (MS) data remain inherently biased as a result of reasons ranging from sample handling to differences caused by the instrumentation. Normalization is the process that aims to account for the bias and make samples more comparable. The selection of a proper normalization method is a pivotal task for the reliability of the downstream analysis and results. Many normalization methods commonly used in proteomics have been adapted from the DNA microarray techniques. Previous studies comparing normalization methods in proteomics have focused mainly on intragroup variation. In this study, several popular and widely used normalization methods representing different strategies in normalization are evaluated using three spike-in and one experimental mouse label-free proteomic data sets. The normalization methods are evaluated in terms of their ability to reduce variation between technical replicates, their effect on differential expression analysis and their effect on the estimation of logarithmic fold changes. Additionally, we examined whether normalizing the whole data globally or in segments for the differential expression analysis has an effect on the performance of the normalization methods. We found that variance stabilization normalization (Vsn) reduced variation the most between technical replicates in all examined data sets. Vsn also performed consistently well in the differential expression analysis. Linear regression normalization and local regression normalization performed also systematically well. Finally, we discuss the choice of a normalization method and some qualities of a suitable normalization method in the light of the results of our evaluation. © The Author 2016. Published by Oxford University Press.

The Spectra Count Label-free Quantitation in Cancer Proteomics

OpenAIRE

Zhou, Weidong; Liotta, Lance A.; Petricoin, Emanuel F.

2012-01-01

Mass spectrometry is used routinely for large-scale protein identification from complex biological mixtures. Recently, relative quantitation approach on the basis of spectra count has been applied in several cancer proteomic studies. In this review, we examine the mechanism of this technique and highlight several important parameters associated with its application.

Hydroponic isotope labeling of entire plants and high-performance mass spectrometry for quantitative plant proteomics.

Science.gov (United States)

Bindschedler, Laurence V; Mills, Davinia J S; Cramer, Rainer

2012-01-01

Hydroponic isotope labeling of entire plants (HILEP) combines hydroponic plant cultivation and metabolic labeling with stable isotopes using (15)N-containing inorganic salts to label whole and mature plants. Employing (15)N salts as the sole nitrogen source for HILEP leads to the production of healthy-looking plants which contain (15)N proteins labeled to nearly 100%. Therefore, HILEP is suitable for quantitative plant proteomic analysis, where plants are grown in either (14)N- or (15)N-hydroponic media and pooled when the biological samples are collected for relative proteome quantitation. The pooled (14)N-/(15)N-protein extracts can be fractionated in any suitable way and digested with a protease for shotgun proteomics, using typically reverse phase liquid chromatography nanoelectrospray ionization tandem mass spectrometry (RPLC-nESI-MS/MS). Best results were obtained with a hybrid ion trap/FT-MS mass spectrometer, combining high mass accuracy and sensitivity for the MS data acquisition with speed and high-throughput MS/MS data acquisition, increasing the number of proteins identified and quantified and improving protein quantitation. Peak processing and picking from raw MS data files, protein identification, and quantitation were performed in a highly automated way using integrated MS data analysis software with minimum manual intervention, thus easing the analytical workflow. In this methodology paper, we describe how to grow Arabidopsis plants hydroponically for isotope labeling using (15)N salts and how to quantitate the resulting proteomes using a convenient workflow that does not require extensive bioinformatics skills.

Mapping in vivo target interaction profiles of covalent inhibitors using chemical proteomics with label-free quantification.

Science.gov (United States)

van Rooden, Eva J; Florea, Bogdan I; Deng, Hui; Baggelaar, Marc P; van Esbroeck, Annelot C M; Zhou, Juan; Overkleeft, Herman S; van der Stelt, Mario

2018-04-01

Activity-based protein profiling (ABPP) has emerged as a valuable chemical proteomics method to guide the therapeutic development of covalent drugs by assessing their on-target engagement and off-target activity. We recently used ABPP to determine the serine hydrolase interaction landscape of the experimental drug BIA 10-2474, thereby providing a potential explanation for the adverse side effects observed with this compound. ABPP allows mapping of protein interaction landscapes of inhibitors in cells, tissues and animal models. Whereas our previous protocol described quantification of proteasome activity using stable-isotope labeling, this protocol describes the procedures for identifying the in vivo selectivity profile of covalent inhibitors with label-free quantitative proteomics. The optimization of our protocol for label-free quantification methods results in high proteome coverage and allows the comparison of multiple biological samples. We demonstrate our protocol by assessing the protein interaction landscape of the diacylglycerol lipase inhibitor DH376 in mouse brain, liver, kidney and testes. The stages of the protocol include tissue lysis, probe incubation, target enrichment, sample preparation, liquid chromatography-mass spectrometry (LC-MS) measurement, data processing and analysis. This approach can be used to study target engagement in a native proteome and to identify potential off targets for the inhibitor under investigation. The entire protocol takes at least 4 d, depending on the number of samples.

Comparison of a Label-Free Quantitative Proteomic Method Based on Peptide Ion Current Area to the Isotope Coded Affinity Tag Method

Directory of Open Access Journals (Sweden)

Young Ah Goo

2008-01-01

Full Text Available Recently, several research groups have published methods for the determination of proteomic expression profiling by mass spectrometry without the use of exogenously added stable isotopes or stable isotope dilution theory. These so-called label-free, methods have the advantage of allowing data on each sample to be acquired independently from all other samples to which they can later be compared in silico for the purpose of measuring changes in protein expression between various biological states. We developed label free software based on direct measurement of peptide ion current area (PICA and compared it to two other methods, a simpler label free method known as spectral counting and the isotope coded affinity tag (ICAT method. Data analysis by these methods of a standard mixture containing proteins of known, but varying, concentrations showed that they performed similarly with a mean squared error of 0.09. Additionally, complex bacterial protein mixtures spiked with known concentrations of standard proteins were analyzed using the PICA label-free method. These results indicated that the PICA method detected all levels of standard spiked proteins at the 90% confidence level in this complex biological sample. This finding confirms that label-free methods, based on direct measurement of the area under a single ion current trace, performed as well as the standard ICAT method. Given the fact that the label-free methods provide ease in experimental design well beyond pair-wise comparison, label-free methods such as our PICA method are well suited for proteomic expression profiling of large numbers of samples as is needed in clinical analysis.

UNiquant, a Program for Quantitative Proteomics Analysis Using Stable Isotope Labeling

Energy Technology Data Exchange (ETDEWEB)

Huang, Xin; Tolmachev, Aleksey V.; Shen, Yulei; Liu, Miao; Huang, Lin; Zhang, Zhixin; Anderson, Gordon A.; Smith, Richard D.; Chan, Wing C.; Hinrichs, Steven; Fu, Kai; Ding, Shi-Jian

2011-03-04

We present UNiquant, a new software program for analyzing stable isotope labeling (SIL) based quantitative proteomics data. UNiquant surpassed the performance of two other platforms, MaxQuant and Mascot Distiller, using complex proteome mixtures having either known or unknown heavy/light ratios. UNiquant is compatible with a broad spectrum of search engines and SIL methods, providing outstanding peptide pair identification and accurate measurement of the relative peptide/protein abundance.

[Methods of quantitative proteomics].

Science.gov (United States)

Kopylov, A T; Zgoda, V G

2007-01-01

In modern science proteomic analysis is inseparable from other fields of systemic biology. Possessing huge resources quantitative proteomics operates colossal information on molecular mechanisms of life. Advances in proteomics help researchers to solve complex problems of cell signaling, posttranslational modification, structure and functional homology of proteins, molecular diagnostics etc. More than 40 various methods have been developed in proteomics for quantitative analysis of proteins. Although each method is unique and has certain advantages and disadvantages all these use various isotope labels (tags). In this review we will consider the most popular and effective methods employing both chemical modifications of proteins and also metabolic and enzymatic methods of isotope labeling.

Assigning Significance in Label-Free Quantitative Proteomics to Include Single-Peptide-Hit Proteins with Low Replicates

OpenAIRE

Li, Qingbo

2010-01-01

When sample replicates are limited in a label-free proteomics experiment, selecting differentially regulated proteins with an assignment of statistical significance remains difficult for proteins with a single-peptide hit or a small fold-change. This paper aims to address this issue. An important component of the approach employed here is to utilize the rule of Minimum number of Permuted Significant Pairings (MPSP) to reduce false positives. The MPSP rule generates permuted sample pairings fr...

Comparative study of label and label-free techniques using shotgun proteomics for relative protein quantification.

Science.gov (United States)

Sjödin, Marcus O D; Wetterhall, Magnus; Kultima, Kim; Artemenko, Konstantin

2013-06-01

The analytical performance of three different strategies, iTRAQ (isobaric tag for relative and absolute quantification), dimethyl labeling (DML) and label free (LF) for relative protein quantification using shotgun proteomics have been evaluated. The methods have been explored using samples containing (i) Bovine proteins in known ratios and (ii) Bovine proteins in known ratios spiked into Escherichia coli. The latter case mimics the actual conditions in a typical biological sample with a few differentially expressed proteins and a bulk of proteins with unchanged ratios. Additionally, the evaluation was performed on both QStar and LTQ-FTICR mass spectrometers. LF LTQ-FTICR was found to have the highest proteome coverage while the highest accuracy based on the artificially regulated proteins was found for DML LTQ-FTICR (54%). A varying linearity (k: 0.55-1.16, r(2): 0.61-0.96) was shown for all methods within selected dynamic ranges. All methods were found to consistently underestimate Bovine protein ratios when matrix proteins were added. However, LF LTQ-FTICR was more tolerant toward a compression effect. A single peptide was demonstrated to be sufficient for a reliable quantification using iTRAQ. A ranking system utilizing several parameters important for quantitative proteomics demonstrated that the overall performance of the five different methods was; DML LTQ-FTICR>iTRAQ QStar>LF LTQ-FTICR>DML QStar>LF QStar. Copyright © 2013 Elsevier B.V. All rights reserved.

Mastitomics, the integrated omics of bovine milk in an experimental model of Streptococcus uberis mastitis: 2. Label-free relative quantitative proteomics.

Science.gov (United States)

Mudaliar, Manikhandan; Tassi, Riccardo; Thomas, Funmilola C; McNeilly, Tom N; Weidt, Stefan K; McLaughlin, Mark; Wilson, David; Burchmore, Richard; Herzyk, Pawel; Eckersall, P David; Zadoks, Ruth N

2016-08-16

Mastitis, inflammation of the mammary gland, is the most common and costly disease of dairy cattle in the western world. It is primarily caused by bacteria, with Streptococcus uberis as one of the most prevalent causative agents. To characterize the proteome during Streptococcus uberis mastitis, an experimentally induced model of intramammary infection was used. Milk whey samples obtained from 6 cows at 6 time points were processed using label-free relative quantitative proteomics. This proteomic analysis complements clinical, bacteriological and immunological studies as well as peptidomic and metabolomic analysis of the same challenge model. A total of 2552 non-redundant bovine peptides were identified, and from these, 570 bovine proteins were quantified. Hierarchical cluster analysis and principal component analysis showed clear clustering of results by stage of infection, with similarities between pre-infection and resolution stages (0 and 312 h post challenge), early infection stages (36 and 42 h post challenge) and late infection stages (57 and 81 h post challenge). Ingenuity pathway analysis identified upregulation of acute phase protein pathways over the course of infection, with dominance of different acute phase proteins at different time points based on differential expression analysis. Antimicrobial peptides, notably cathelicidins and peptidoglycan recognition protein, were upregulated at all time points post challenge and peaked at 57 h, which coincided with 10 000-fold decrease in average bacterial counts. The integration of clinical, bacteriological, immunological and quantitative proteomics and other-omic data provides a more detailed systems level view of the host response to mastitis than has been achieved previously.

Identification of indicator proteins associated with flooding injury in soybean seedlings using label-free quantitative proteomics.

Science.gov (United States)

Nanjo, Yohei; Nakamura, Takuji; Komatsu, Setsuko

2013-11-01

Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. Dissolved oxygen levels in the floodwater were decreased by the seedlings and correlated with the degree of injury. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. The analysis showed that the abundance of proteins involved in cell wall modification, such as polygalacturonase inhibitor-like and expansin-like B1-like proteins, which may be associated with the defense system, increased dependence on stress at both the protein and mRNA levels in all organs during flooding. The manner of alteration in abundance of these proteins was distinct from those of other responsive proteins. Furthermore, proteins also showing specific changes in abundance in the root tip included protein phosphatase 2A subunit-like proteins, which are possibly involved in flooding-induced root tip cell death. Additionally, decreases in abundance of cell wall synthesis-related proteins, such as cinnamyl-alcohol dehydrogenase and cellulose synthase-interactive protein-like proteins, were identified in hypocotyls of seedlings grown for 3 days after flooding, and these proteins may be associated with suppression of growth after flooding. These flooding injury-associated proteins can be defined as indicator proteins for severity of flooding stress in soybean.

The proteome of human liver peroxisomes: identification of five new peroxisomal constituents by a label-free quantitative proteomics survey.

Directory of Open Access Journals (Sweden)

Thomas Gronemeyer

Full Text Available The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. A label-free quantitative study of 314 proteins across the density gradient was accomplished using high resolution mass spectrometry. By pairing statistical data evaluation, cDNA cloning and in vivo colocalization studies, we report the association of five new proteins with human liver peroxisomes. Among these, isochorismatase domain containing 1 protein points to the existence of a new metabolic pathway and hydroxysteroid dehydrogenase like 2 protein is likely involved in the transport or β-oxidation of fatty acids in human peroxisomes. The detection of alcohol dehydrogenase 1A suggests the presence of an alternative alcohol-oxidizing system in hepatic peroxisomes. In addition, lactate dehydrogenase A and malate dehydrogenase 1 partially associate with human liver peroxisomes and enzyme activity profiles support the idea that NAD(+ becomes regenerated during fatty acid β-oxidation by alternative shuttling processes in human peroxisomes involving lactate dehydrogenase and/or malate dehydrogenase. Taken together, our data represent a valuable resource for future studies of peroxisome biochemistry that will advance research of human peroxisomes in health and disease.

Analytical performance of reciprocal isotope labeling of proteome digests for quantitative proteomics and its application for comparative studies of aerobic and anaerobic Escherichia coli proteomes

International Nuclear Information System (INIS)

Lo, Andy; Weiner, Joel H.; Li, Liang

2013-01-01

Graphical abstract: -- Highlights: •Investigating a strategy of reciprocal isotope labeling of comparative samples. •Filtering out incorrect peptide identification or quantification values. •Analyzing the proteome changes of E. coli cells grown aerobically or anaerobically. •Presenting guidelines for reciprocal labeling experimental design. -- Abstract: Due to limited sample amounts, instrument time considerations, and reagent costs, only a small number of replicate experiments are typically performed for quantitative proteome analyses. Generation of reproducible data that can be readily assessed for consistency within a small number of datasets is critical for accurate quantification. We report our investigation of a strategy using reciprocal isotope labeling of two comparative samples as a tool for determining proteome changes. Reciprocal labeling was evaluated to determine the internal consistency of quantified proteome changes from Escherichia coli grown under aerobic and anaerobic conditions. Qualitatively, the peptide overlap between replicate analyses of the same sample and reverse labeled samples were found to be within 8%. Quantitatively, reciprocal analyses showed only a slight increase in average overall inconsistency when compared with replicate analyses (1.29 vs. 1.24-fold difference). Most importantly, reverse labeling was successfully used to identify spurious values resulting from incorrect peptide identifications and poor peak fitting. After removal of 5% of the peptide data with low reproducibility, a total of 275 differentially expressed proteins (>1.50-fold difference) were consistently identified and were then subjected to bioinformatics analysis. General considerations and guidelines for reciprocal labeling experimental design and biological significance of obtained results are discussed

Identification of Host Defense-Related Proteins Using Label-Free Quantitative Proteomic Analysis of Milk Whey from Cows with Staphylococcus aureus Subclinical Mastitis

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Shaimaa Abdelmegid

2017-12-01

Full Text Available Staphylococcus aureus is the most common contagious pathogen associated with bovine subclinical mastitis. Current diagnosis of S. aureus mastitis is based on bacteriological culture of milk samples and somatic cell counts, which lack either sensitivity or specificity. Identification of milk proteins that contribute to host defense and their variable responses to pathogenic stimuli would enable the characterization of putative biomarkers of subclinical mastitis. To accomplish this, milk whey samples from healthy and mastitic dairy cows were analyzed using a label-free quantitative proteomics approach. In total, 90 proteins were identified, of which 25 showed significant differential abundance between healthy and mastitic samples. In silico functional analyses indicated the involvement of the differentially abundant proteins in biological mechanisms and signaling pathways related to host defense including pathogen-recognition, direct antimicrobial function, and the acute-phase response. This proteomics and bioinformatics analysis not only facilitates the identification of putative biomarkers of S. aureus subclinical mastitis but also recapitulates previous findings demonstrating the abundance of host defense proteins in intramammary infection. All mass spectrometry data are available via ProteomeXchange with identifier PXD007516.

A comprehensive evaluation of popular proteomics software workflows for label-free proteome quantification and imputation.

Science.gov (United States)

Välikangas, Tommi; Suomi, Tomi; Elo, Laura L

2017-05-31

Label-free mass spectrometry (MS) has developed into an important tool applied in various fields of biological and life sciences. Several software exist to process the raw MS data into quantified protein abundances, including open source and commercial solutions. Each software includes a set of unique algorithms for different tasks of the MS data processing workflow. While many of these algorithms have been compared separately, a thorough and systematic evaluation of their overall performance is missing. Moreover, systematic information is lacking about the amount of missing values produced by the different proteomics software and the capabilities of different data imputation methods to account for them.In this study, we evaluated the performance of five popular quantitative label-free proteomics software workflows using four different spike-in data sets. Our extensive testing included the number of proteins quantified and the number of missing values produced by each workflow, the accuracy of detecting differential expression and logarithmic fold change and the effect of different imputation and filtering methods on the differential expression results. We found that the Progenesis software performed consistently well in the differential expression analysis and produced few missing values. The missing values produced by the other software decreased their performance, but this difference could be mitigated using proper data filtering or imputation methods. Among the imputation methods, we found that the local least squares (lls) regression imputation consistently increased the performance of the software in the differential expression analysis, and a combination of both data filtering and local least squares imputation increased performance the most in the tested data sets. © The Author 2017. Published by Oxford University Press.

Label-Free Quantitative Proteomic Analysis of Chitosan Oligosaccharide-Treated Rice Infected with Southern Rice Black-Streaked Dwarf Virus.

Science.gov (United States)

Yang, Anming; Yu, Lu; Chen, Zhuo; Zhang, Shanxue; Shi, Jing; Zhao, Xiaozhen; Yang, Yuanyou; Hu, Deyu; Song, Baoan

2017-05-18

Southern rice black-streaked dwarf virus (SRBSDV) has spread from thesouth of China to the north of Vietnam in the past few years and severelyinfluenced rice production. Its long incubation period and early symptoms are not evident; thus, controlling it is difficult. Chitosan oligosaccharide (COS) is a green plant immunomodulator. Early studies showed that preventing and controlling SRBSDV have a certain effect and reduce disease infection rate, but its underlying controlling and preventing mechanism is unclear. In this study, label-free proteomics was used to analyze differentially expressed proteins in rice after COS treatment. The results showed that COS can up-regulate the plant defense-related proteins and down-regulate the protein expression levels of SRBSDV. Meanwhile, quantitative real-time PCR test results showed that COS can improve defense gene expression in rice. Moreover, COS can enhance the defense enzymatic activities of peroxidase, superoxide dismutase and catalase through mitogen-activated protein kinase signaling cascade pathway, and enhance the rice disease resistance.

EBprot: Statistical analysis of labeling-based quantitative proteomics data.

Science.gov (United States)

Koh, Hiromi W L; Swa, Hannah L F; Fermin, Damian; Ler, Siok Ghee; Gunaratne, Jayantha; Choi, Hyungwon

2015-08-01

Labeling-based proteomics is a powerful method for detection of differentially expressed proteins (DEPs). The current data analysis platform typically relies on protein-level ratios, which is obtained by summarizing peptide-level ratios for each protein. In shotgun proteomics, however, some proteins are quantified with more peptides than others, and this reproducibility information is not incorporated into the differential expression (DE) analysis. Here, we propose a novel probabilistic framework EBprot that directly models the peptide-protein hierarchy and rewards the proteins with reproducible evidence of DE over multiple peptides. To evaluate its performance with known DE states, we conducted a simulation study to show that the peptide-level analysis of EBprot provides better receiver-operating characteristic and more accurate estimation of the false discovery rates than the methods based on protein-level ratios. We also demonstrate superior classification performance of peptide-level EBprot analysis in a spike-in dataset. To illustrate the wide applicability of EBprot in different experimental designs, we applied EBprot to a dataset for lung cancer subtype analysis with biological replicates and another dataset for time course phosphoproteome analysis of EGF-stimulated HeLa cells with multiplexed labeling. Through these examples, we show that the peptide-level analysis of EBprot is a robust alternative to the existing statistical methods for the DE analysis of labeling-based quantitative datasets. The software suite is freely available on the Sourceforge website http://ebprot.sourceforge.net/. All MS data have been deposited in the ProteomeXchange with identifier PXD001426 (http://proteomecentral.proteomexchange.org/dataset/PXD001426/). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Automated selected reaction monitoring software for accurate label-free protein quantification.

Science.gov (United States)

Teleman, Johan; Karlsson, Christofer; Waldemarson, Sofia; Hansson, Karin; James, Peter; Malmström, Johan; Levander, Fredrik

2012-07-06

Selected reaction monitoring (SRM) is a mass spectrometry method with documented ability to quantify proteins accurately and reproducibly using labeled reference peptides. However, the use of labeled reference peptides becomes impractical if large numbers of peptides are targeted and when high flexibility is desired when selecting peptides. We have developed a label-free quantitative SRM workflow that relies on a new automated algorithm, Anubis, for accurate peak detection. Anubis efficiently removes interfering signals from contaminating peptides to estimate the true signal of the targeted peptides. We evaluated the algorithm on a published multisite data set and achieved results in line with manual data analysis. In complex peptide mixtures from whole proteome digests of Streptococcus pyogenes we achieved a technical variability across the entire proteome abundance range of 6.5-19.2%, which was considerably below the total variation across biological samples. Our results show that the label-free SRM workflow with automated data analysis is feasible for large-scale biological studies, opening up new possibilities for quantitative proteomics and systems biology.

Quantitative proteome profiling of normal human circulating microparticles

DEFF Research Database (Denmark)

Østergaard, Ole; Nielsen, Christoffer T; Iversen, Line V

2012-01-01

Circulating microparticles (MPs) are produced as part of normal physiology. Their numbers, origin, and composition change in pathology. Despite this, the normal MP proteome has not yet been characterized with standardized high-resolution methods. We here quantitatively profile the normal MP...... proteome using nano-LC-MS/MS on an LTQ-Orbitrap with optimized sample collection, preparation, and analysis of 12 different normal samples. Analytical and procedural variation were estimated in triply processed samples analyzed in triplicate from two different donors. Label-free quantitation was validated...... by the correlation of cytoskeletal protein intensities with MP numbers obtained by flow cytometry. Finally, the validity of using pooled samples was evaluated using overlap protein identification numbers and multivariate data analysis. Using conservative parameters, 536 different unique proteins were quantitated...

A flexible statistical model for alignment of label-free proteomics data--incorporating ion mobility and product ion information.

Science.gov (United States)

Benjamin, Ashlee M; Thompson, J Will; Soderblom, Erik J; Geromanos, Scott J; Henao, Ricardo; Kraus, Virginia B; Moseley, M Arthur; Lucas, Joseph E

2013-12-16

The goal of many proteomics experiments is to determine the abundance of proteins in biological samples, and the variation thereof in various physiological conditions. High-throughput quantitative proteomics, specifically label-free LC-MS/MS, allows rapid measurement of thousands of proteins, enabling large-scale studies of various biological systems. Prior to analyzing these information-rich datasets, raw data must undergo several computational processing steps. We present a method to address one of the essential steps in proteomics data processing--the matching of peptide measurements across samples. We describe a novel method for label-free proteomics data alignment with the ability to incorporate previously unused aspects of the data, particularly ion mobility drift times and product ion information. We compare the results of our alignment method to PEPPeR and OpenMS, and compare alignment accuracy achieved by different versions of our method utilizing various data characteristics. Our method results in increased match recall rates and similar or improved mismatch rates compared to PEPPeR and OpenMS feature-based alignment. We also show that the inclusion of drift time and product ion information results in higher recall rates and more confident matches, without increases in error rates. Based on the results presented here, we argue that the incorporation of ion mobility drift time and product ion information are worthy pursuits. Alignment methods should be flexible enough to utilize all available data, particularly with recent advancements in experimental separation methods.

Quantitative label-free proteomics for discovery of biomarkers in cerebrospinal fluid: assessment of technical and inter-individual variation.

Directory of Open Access Journals (Sweden)

Richard J Perrin

Full Text Available Biomarkers are required for pre-symptomatic diagnosis, treatment, and monitoring of neurodegenerative diseases such as Alzheimer's disease. Cerebrospinal fluid (CSF is a favored source because its proteome reflects the composition of the brain. Ideal biomarkers have low technical and inter-individual variability (subject variance among control subjects to minimize overlaps between clinical groups. This study evaluates a process of multi-affinity fractionation (MAF and quantitative label-free liquid chromatography tandem mass spectrometry (LC-MS/MS for CSF biomarker discovery by (1 identifying reparable sources of technical variability, (2 assessing subject variance and residual technical variability for numerous CSF proteins, and (3 testing its ability to segregate samples on the basis of desired biomarker characteristics.Fourteen aliquots of pooled CSF and two aliquots from six cognitively normal individuals were randomized, enriched for low-abundance proteins by MAF, digested endoproteolytically, randomized again, and analyzed by nano-LC-MS. Nano-LC-MS data were time and m/z aligned across samples for relative peptide quantification. Among 11,433 aligned charge groups, 1360 relatively abundant ones were annotated by MS2, yielding 823 unique peptides. Analyses, including Pearson correlations of annotated LC-MS ion chromatograms, performed for all pairwise sample comparisons, identified several sources of technical variability: i incomplete MAF and keratins; ii globally- or segmentally-decreased ion current in isolated LC-MS analyses; and iii oxidized methionine-containing peptides. Exclusion of these sources yielded 609 peptides representing 81 proteins. Most of these proteins showed very low coefficients of variation (CV<5% whether they were quantified from the mean of all or only the 2 most-abundant peptides. Unsupervised clustering, using only 24 proteins selected for high subject variance, yielded perfect segregation of pooled and

The beauty of being (label)-free: sample preparation methods for SWATH-MS and next-generation targeted proteomics

Science.gov (United States)

Campbell, Kate; Deery, Michael J.; Lilley, Kathryn S.; Ralser, Markus

2014-01-01

The combination of qualitative analysis with label-free quantification has greatly facilitated the throughput and flexibility of novel proteomic techniques. However, such methods rely heavily on robust and reproducible sample preparation procedures. Here, we benchmark a selection of in gel, on filter, and in solution digestion workflows for their application in label-free proteomics. Each procedure was associated with differing advantages and disadvantages. The in gel methods interrogated were cost effective, but were limited in throughput and digest efficiency. Filter-aided sample preparations facilitated reasonable processing times and yielded a balanced representation of membrane proteins, but led to a high signal variation in quantification experiments. Two in solution digest protocols, however, gave optimal performance for label-free proteomics. A protocol based on the detergent RapiGest led to the highest number of detected proteins at second-best signal stability, while a protocol based on acetonitrile-digestion, RapidACN, scored best in throughput and signal stability but came second in protein identification. In addition, we compared label-free data dependent (DDA) and data independent (SWATH) acquisition on a TripleTOF 5600 instrument. While largely similar in protein detection, SWATH outperformed DDA in quantification, reducing signal variation and markedly increasing the number of precisely quantified peptides. PMID:24741437

Quantitative Proteomics Reveals Temporal Proteomic Changes in Signaling Pathways during BV2 Mouse Microglial Cell Activation.

Science.gov (United States)

Woo, Jongmin; Han, Dohyun; Wang, Joseph Injae; Park, Joonho; Kim, Hyunsoo; Kim, Youngsoo

2017-09-01

The development of systematic proteomic quantification techniques in systems biology research has enabled one to perform an in-depth analysis of cellular systems. We have developed a systematic proteomic approach that encompasses the spectrum from global to targeted analysis on a single platform. We have applied this technique to an activated microglia cell system to examine changes in the intracellular and extracellular proteomes. Microglia become activated when their homeostatic microenvironment is disrupted. There are varying degrees of microglial activation, and we chose to focus on the proinflammatory reactive state that is induced by exposure to such stimuli as lipopolysaccharide (LPS) and interferon-gamma (IFN-γ). Using an improved shotgun proteomics approach, we identified 5497 proteins in the whole-cell proteome and 4938 proteins in the secretome that were associated with the activation of BV2 mouse microglia by LPS or IFN-γ. Of the differentially expressed proteins in stimulated microglia, we classified pathways that were related to immune-inflammatory responses and metabolism. Our label-free parallel reaction monitoring (PRM) approach made it possible to comprehensively measure the hyper-multiplex quantitative value of each protein by high-resolution mass spectrometry. Over 450 peptides that corresponded to pathway proteins and direct or indirect interactors via the STRING database were quantified by label-free PRM in a single run. Moreover, we performed a longitudinal quantification of secreted proteins during microglial activation, in which neurotoxic molecules that mediate neuronal cell loss in the brain are released. These data suggest that latent pathways that are associated with neurodegenerative diseases can be discovered by constructing and analyzing a pathway network model of proteins. Furthermore, this systematic quantification platform has tremendous potential for applications in large-scale targeted analyses. The proteomics data for

LFQProfiler and RNP(xl): Open-Source Tools for Label-Free Quantification and Protein-RNA Cross-Linking Integrated into Proteome Discoverer.

Science.gov (United States)

Veit, Johannes; Sachsenberg, Timo; Chernev, Aleksandar; Aicheler, Fabian; Urlaub, Henning; Kohlbacher, Oliver

2016-09-02

Modern mass spectrometry setups used in today's proteomics studies generate vast amounts of raw data, calling for highly efficient data processing and analysis tools. Software for analyzing these data is either monolithic (easy to use, but sometimes too rigid) or workflow-driven (easy to customize, but sometimes complex). Thermo Proteome Discoverer (PD) is a powerful software for workflow-driven data analysis in proteomics which, in our eyes, achieves a good trade-off between flexibility and usability. Here, we present two open-source plugins for PD providing additional functionality: LFQProfiler for label-free quantification of peptides and proteins, and RNP(xl) for UV-induced peptide-RNA cross-linking data analysis. LFQProfiler interacts with existing PD nodes for peptide identification and validation and takes care of the entire quantitative part of the workflow. We show that it performs at least on par with other state-of-the-art software solutions for label-free quantification in a recently published benchmark ( Ramus, C.; J. Proteomics 2016 , 132 , 51 - 62 ). The second workflow, RNP(xl), represents the first software solution to date for identification of peptide-RNA cross-links including automatic localization of the cross-links at amino acid resolution and localization scoring. It comes with a customized integrated cross-link fragment spectrum viewer for convenient manual inspection and validation of the results.

Gel-free/label-free proteomic, photosynthetic, and biochemical analysis of cowpea (Vigna unguiculata [L.] Walp.) resistance against Cowpea severe mosaic virus (CPSMV).

Science.gov (United States)

Varela, Anna Lidia N; Komatsu, Setsuko; Wang, Xin; Silva, Rodolpho G G; Souza, Pedro Filho N; Lobo, Ana Karla M; Vasconcelos, Ilka M; Silveira, Joaquim A G; Oliveira, Jose T A

2017-06-23

Cowpea severe mosaic virus (CPSMV) causes significant losses in cowpea (Vigna unguiculata) production. In this present study biochemical, physiological, and proteomic analysis were done to identify pathways and defense proteins that are altered during the incompatible interaction between the cowpea genotype BRS-Marataoã and CPSMV. The leaf protein extracts from mock- (MI) and CPSMV-inoculated plantlets (V) were evaluated at 2 and 6days post-inoculation (DPI). Data support the assumptions that increases in biochemical (high hydrogen peroxide, antioxidant enzymes, and secondary compounds) and physiological responses (high photosynthesis index and chlorophyll content), confirmed by label-free comparative proteomic approach, in which quantitative changes in proteasome proteins, proteins related to photosynthesis, redox homeostasis, regulation factors/RNA processing proteins were observed may be implicated in the resistance of BRS-Marataoã to CPSMV. This pioneering study provides information for the selection of specific pathways and proteins, altered in this incompatible relationship, which could be chosen as targets for detailed studies to advance our understanding of the molecular, physiological, and biochemistry basis of the resistance mechanism of cowpea and design approachs to engineer plants that are more productive. This is a pioneering study in which an incompatible relationship between a resistant cowpea and Cowpea severe mosaic virus (CPSMV) was conducted to comparatively evaluate proteomic profiles by Gel-free/label-free methodology and some physiological and biochemical parameters to shed light on how a resistant cowpea cultivar deals with the virus attack. Specific proteins and associated pathways were altered in the cowpea plants challenged with CPSMV and will contribute to our knowledge on the biological process tailored by cowpea in response to CPSMV. Copyright © 2017 Elsevier B.V. All rights reserved.

Guidelines for reporting quantitative mass spectrometry based experiments in proteomics.

Science.gov (United States)

Martínez-Bartolomé, Salvador; Deutsch, Eric W; Binz, Pierre-Alain; Jones, Andrew R; Eisenacher, Martin; Mayer, Gerhard; Campos, Alex; Canals, Francesc; Bech-Serra, Joan-Josep; Carrascal, Montserrat; Gay, Marina; Paradela, Alberto; Navajas, Rosana; Marcilla, Miguel; Hernáez, María Luisa; Gutiérrez-Blázquez, María Dolores; Velarde, Luis Felipe Clemente; Aloria, Kerman; Beaskoetxea, Jabier; Medina-Aunon, J Alberto; Albar, Juan P

2013-12-16

Mass spectrometry is already a well-established protein identification tool and recent methodological and technological developments have also made possible the extraction of quantitative data of protein abundance in large-scale studies. Several strategies for absolute and relative quantitative proteomics and the statistical assessment of quantifications are possible, each having specific measurements and therefore, different data analysis workflows. The guidelines for Mass Spectrometry Quantification allow the description of a wide range of quantitative approaches, including labeled and label-free techniques and also targeted approaches such as Selected Reaction Monitoring (SRM). The HUPO Proteomics Standards Initiative (HUPO-PSI) has invested considerable efforts to improve the standardization of proteomics data handling, representation and sharing through the development of data standards, reporting guidelines, controlled vocabularies and tooling. In this manuscript, we describe a key output from the HUPO-PSI-namely the MIAPE Quant guidelines, which have developed in parallel with the corresponding data exchange format mzQuantML [1]. The MIAPE Quant guidelines describe the HUPO-PSI proposal concerning the minimum information to be reported when a quantitative data set, derived from mass spectrometry (MS), is submitted to a database or as supplementary information to a journal. The guidelines have been developed with input from a broad spectrum of stakeholders in the proteomics field to represent a true consensus view of the most important data types and metadata, required for a quantitative experiment to be analyzed critically or a data analysis pipeline to be reproduced. It is anticipated that they will influence or be directly adopted as part of journal guidelines for publication and by public proteomics databases and thus may have an impact on proteomics laboratories across the world. This article is part of a Special Issue entitled: Standardization and

Label-free protein profiling of formalin-fixed paraffin-embedded (FFPE) heart tissue reveals immediate mitochondrial impairment after ionising radiation.

Science.gov (United States)

Azimzadeh, Omid; Scherthan, Harry; Yentrapalli, Ramesh; Barjaktarovic, Zarko; Ueffing, Marius; Conrad, Marcus; Neff, Frauke; Calzada-Wack, Julia; Aubele, Michaela; Buske, Christian; Atkinson, Michael J; Hauck, Stefanie M; Tapio, Soile

2012-04-18

Qualitative proteome profiling of formalin-fixed, paraffin-embedded (FFPE) tissue is advancing the field of clinical proteomics. However, quantitative proteome analysis of FFPE tissue is hampered by the lack of an efficient labelling method. The usage of conventional protein labelling on FFPE tissue has turned out to be inefficient. Classical labelling targets lysine residues that are blocked by the formalin treatment. The aim of this study was to establish a quantitative proteomics analysis of FFPE tissue by combining the label-free approach with optimised protein extraction and separation conditions. As a model system we used FFPE heart tissue of control and exposed C57BL/6 mice after total body irradiation using a gamma ray dose of 3 gray. We identified 32 deregulated proteins (p≤0.05) in irradiated hearts 24h after the exposure. The proteomics data were further evaluated and validated by bioinformatics and immunoblotting investigation. In good agreement with our previous results using fresh-frozen tissue, the analysis indicated radiation-induced alterations in three main biological pathways: respiratory chain, lipid metabolism and pyruvate metabolism. The label-free approach enables the quantitative measurement of radiation-induced alterations in FFPE tissue and facilitates retrospective biomarker identification using clinical archives. Copyright © 2012 Elsevier B.V. All rights reserved.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) Applied to Quantitative Proteomics of Bacillus subtilis

DEFF Research Database (Denmark)

Soufi, Boumediene; Kumar, C.; Gnad, F.

2010-01-01

We applied stable isotope labeling by amino acids in cell culture (SILAC) to large-scale quantitative proteomics analyses of the model bacterium Bacillus subtilis in two physiological conditions: growth on succinate and growth under phosphate starvation. Using a B. subtilis strain auxotrophic...... of the most comprehensive quantitative proteomics studies in bacteria, covering more than 75% of the B. subtilis genes expressed in the log phase of growth. Furthermore, we detect and quantify dynamics of 35 Ser/Thr/Tyr phosphorylation sites under growth on succinate, and 10 phosphorylation sites under...

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

Directory of Open Access Journals (Sweden)

Saeed Alexander I

2008-12-01

Full Text Available Abstract Background Mass spectrometry (MS based label-free protein quantitation has mainly focused on analysis of ion peak heights and peptide spectral counts. Most analyses of tandem mass spectrometry (MS/MS data begin with an enzymatic digestion of a complex protein mixture to generate smaller peptides that can be separated and identified by an MS/MS instrument. Peptide spectral counting techniques attempt to quantify protein abundance by counting the number of detected tryptic peptides and their corresponding MS spectra. However, spectral counting is confounded by the fact that peptide physicochemical properties severely affect MS detection resulting in each peptide having a different detection probability. Lu et al. (2007 described a modified spectral counting technique, Absolute Protein Expression (APEX, which improves on basic spectral counting methods by including a correction factor for each protein (called Oi value that accounts for variable peptide detection by MS techniques. The technique uses machine learning classification to derive peptide detection probabilities that are used to predict the number of tryptic peptides expected to be detected for one molecule of a particular protein (Oi. This predicted spectral count is compared to the protein's observed MS total spectral count during APEX computation of protein abundances. Results The APEX Quantitative Proteomics Tool, introduced here, is a free open source Java application that supports the APEX protein quantitation technique. The APEX tool uses data from standard tandem mass spectrometry proteomics experiments and provides computational support for APEX protein abundance quantitation through a set of graphical user interfaces that partition thparameter controls for the various processing tasks. The tool also provides a Z-score analysis for identification of significant differential protein expression, a utility to assess APEX classifier performance via cross validation, and a

The goat (Capra hircus) mammary gland secretory tissue proteome as influenced by weight loss: A study using label free proteomics

DEFF Research Database (Denmark)

Hernandez Castellano, Lorenzo E; Ferreira, Ana M.; Nanni, Paolo

2016-01-01

Seasonal weight loss (SWL) is a significant limitation to animal production. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Herein, label free proteomics was used to characterize the effects of SWL in two goat breeds with different levels of adaptatio...

Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

Science.gov (United States)

Quecine, Maria Carolina; Leite, Thiago Falda; Bini, Andressa Peres; Regiani, Thais; Franceschini, Lívia Maria; Budzinski, Ilara Gabriela Frasson; Marques, Felipe Garbelini; Labate, Mônica Teresa Veneziano; Guidetti-Gonzalez, Simone; Moon, David Henry; Labate, Carlos Alberto

2016-01-01

Puccinia psidii sensu lato (s.l.) is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava) and eucalyptus leaves (PpEucalyptus). NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database) resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

Label-Free Quantitative Proteomic Analysis of Puccinia psidii Uredospores Reveals Differences of Fungal Populations Infecting Eucalyptus and Guava.

Directory of Open Access Journals (Sweden)

Maria Carolina Quecine

Full Text Available Puccinia psidii sensu lato (s.l. is the causal agent of eucalyptus and guava rust, but it also attacks a wide range of plant species from the myrtle family, resulting in a significant genetic and physiological variability among populations accessed from different hosts. The uredospores are crucial to P. psidii dissemination in the field. Although they are important for the fungal pathogenesis, their molecular characterization has been poorly studied. In this work, we report the first in-depth proteomic analysis of P. psidii s.l. uredospores from two contrasting populations: guava fruits (PpGuava and eucalyptus leaves (PpEucalyptus. NanoUPLC-MSE was used to generate peptide spectra that were matched to the UniProt Puccinia genera sequences (UniProt database resulting in the first proteomic analysis of the phytopathogenic fungus P. psidii. Three hundred and fourty proteins were detected and quantified using Label free proteomics. A significant number of unique proteins were found for each sample, others were significantly more or less abundant, according to the fungal populations. In PpGuava population, many proteins correlated with fungal virulence, such as malate dehydrogenase, proteossomes subunits, enolases and others were increased. On the other hand, PpEucalyptus proteins involved in biogenesis, protein folding and translocation were increased, supporting the physiological variability of the fungal populations according to their protein reservoirs and specific host interaction strategies.

Target identification of natural and traditional medicines with quantitative chemical proteomics approaches.

Science.gov (United States)

Wang, Jigang; Gao, Liqian; Lee, Yew Mun; Kalesh, Karunakaran A; Ong, Yong Siang; Lim, Jaehong; Jee, Joo-Eun; Sun, Hongyan; Lee, Su Seong; Hua, Zi-Chun; Lin, Qingsong

2016-06-01

Natural and traditional medicines, being a great source of drugs and drug leads, have regained wide interests due to the limited success of high-throughput screening of compound libraries in the past few decades and the recent technology advancement. Many drugs/bioactive compounds exert their functions through interaction with their protein targets, with more and more drugs showing their ability to target multiple proteins, thus target identification has an important role in drug discovery and biomedical research fields. Identifying drug targets not only furthers the understanding of the mechanism of action (MOA) of a drug but also reveals its potential therapeutic applications and adverse side effects. Chemical proteomics makes use of affinity chromatography approaches coupled with mass spectrometry to systematically identify small molecule-protein interactions. Although traditional affinity-based chemical proteomics approaches have made great progress in the identification of cellular targets and elucidation of MOAs of many bioactive molecules, nonspecific binding remains a major issue which may reduce the accuracy of target identification and may hamper the drug development process. Recently, quantitative proteomics approaches, namely, metabolic labeling, chemical labeling, or label-free approaches, have been implemented in target identification to overcome such limitations. In this review, we will summarize and discuss the recent advances in the application of various quantitative chemical proteomics approaches for the identification of targets of natural and traditional medicines. Copyright © 2016. Published by Elsevier Inc.

Comparative analysis of Brassica napus plasma membrane proteins under phosphorus deficiency using label-free and MaxQuant-based proteomics approaches.

Science.gov (United States)

Chen, Shuisen; Luo, Ying; Ding, Guangda; Xu, Fangsen

2016-02-05

Phosphorus (P) deficiency is a primary constraint for plant growth in terrestrial ecosystems. To better understand the genotypic differences in the adaptation mechanism of Brassica napus to P deficiency, we purified the plasma membrane (PM) from the roots of two genotypes: P-efficient "Eyou Changjia" and P-inefficient "B104-2". Combining label-free quantitative proteomics with the MaxQuant approach, a total of 71 proteins that significantly changed in abundances were identified in the two genotypes in response to P-free starvation, including 31 in "Eyou Changjia" and 40 in "B104-2". Based on comparative genomics study, 28 proteins were mapped to the confidence intervals of quantitative trait loci (QTLs) for P efficiency related traits. Seven decreased proteins with transporter activity were found to be located in the PM by subcellular localization analyses. These proteins involved in intracellular protein transport and ATP hydrolysis coupled proton transport were mapped to the QTL for P content and dry weight. Compared with "B104-2", more decreased proteins referring to transporter activity were found in "Eyou Changjia", showing that substance exchange was decreased in response to short-term P-free starvation. Together with the finding, more decreased proteins functioning in signal transduction and protein synthesis/degradation suggested that "Eyou Changjia" could slow the progression of growth and save more P in response to short-term P-free starvation. P deficiency seriously limits the production and quality of B. napus. Roots absorb water and nutrients and anchor the plant in the soil. Therefore, to study root PM proteome under P stress would be helpful to understand the adaptation mechanism for P deficiency. However, PM proteome analysis in B. napus has been seldom reported due to the high hydrophobicity and low abundance of PM. Thus, we herein investigated the PM proteome alteration of roots in two B. napus genotypes, with different P deficient tolerances, in

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

KAUST Repository

Komatsu, Setsuko

2017-05-10

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database.

Science.gov (United States)

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-06-23

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max 'Enrei'). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. The Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all predicted proteins from

Integration of gel-based and gel-free proteomic data for functional analysis of proteins through Soybean Proteome Database

KAUST Repository

Komatsu, Setsuko; Wang, Xin; Yin, Xiaojian; Nanjo, Yohei; Ohyanagi, Hajime; Sakata, Katsumi

2017-01-01

The Soybean Proteome Database (SPD) stores data on soybean proteins obtained with gel-based and gel-free proteomic techniques. The database was constructed to provide information on proteins for functional analyses. The majority of the data is focused on soybean (Glycine max ‘Enrei’). The growth and yield of soybean are strongly affected by environmental stresses such as flooding. The database was originally constructed using data on soybean proteins separated by two-dimensional polyacrylamide gel electrophoresis, which is a gel-based proteomic technique. Since 2015, the database has been expanded to incorporate data obtained by label-free mass spectrometry-based quantitative proteomics, which is a gel-free proteomic technique. Here, the portions of the database consisting of gel-free proteomic data are described. The gel-free proteomic database contains 39,212 proteins identified in 63 sample sets, such as temporal and organ-specific samples of soybean plants grown under flooding stress or non-stressed conditions. In addition, data on organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored. Furthermore, the database integrates multiple omics data such as genomics, transcriptomics, metabolomics, and proteomics. The SPD database is accessible at http://proteome.dc.affrc.go.jp/Soybean/. Biological significanceThe Soybean Proteome Database stores data obtained from both gel-based and gel-free proteomic techniques. The gel-free proteomic database comprises 39,212 proteins identified in 63 sample sets, such as different organs of soybean plants grown under flooding stress or non-stressed conditions in a time-dependent manner. In addition, organellar proteins identified in mitochondria, nuclei, and endoplasmic reticulum are stored in the gel-free proteomics database. A total of 44,704 proteins, including 5490 proteins identified using a gel-based proteomic technique, are stored in the SPD. It accounts for approximately 80% of all

Differential quantitative proteomics of Porphyromonas gingivalis by linear ion trap mass spectrometry: Non-label methods comparison, q-values and LOWESS curve fitting

Science.gov (United States)

Xia, Qiangwei; Wang, Tiansong; Park, Yoonsuk; Lamont, Richard J.; Hackett, Murray

2007-01-01

Differential analysis of whole cell proteomes by mass spectrometry has largely been applied using various forms of stable isotope labeling. While metabolic stable isotope labeling has been the method of choice, it is often not possible to apply such an approach. Four different label free ways of calculating expression ratios in a classic "two-state" experiment are compared: signal intensity at the peptide level, signal intensity at the protein level, spectral counting at the peptide level, and spectral counting at the protein level. The quantitative data were mined from a dataset of 1245 qualitatively identified proteins, about 56% of the protein encoding open reading frames from Porphyromonas gingivalis, a Gram-negative intracellular pathogen being studied under extracellular and intracellular conditions. Two different control populations were compared against P. gingivalis internalized within a model human target cell line. The q-value statistic, a measure of false discovery rate previously applied to transcription microarrays, was applied to proteomics data. For spectral counting, the most logically consistent estimate of random error came from applying the locally weighted scatter plot smoothing procedure (LOWESS) to the most extreme ratios generated from a control technical replicate, thus setting upper and lower bounds for the region of experimentally observed random error.

Prediction of skin anti-aging clinical benefits of an association of ingredients from marine and maritime origins: Ex vivo evaluation using a label-free quantitative proteomic and customized data processing approach.

Science.gov (United States)

Hameury, Sebastien; Borderie, Laurent; Monneuse, Jean-Marc; Skorski, Gilbert; Pradines, Dominique

2018-05-23

The application of ingredients from marine and maritime origins is increasingly common in skin care products, driven by consumer expectations for natural ingredients. However, these ingredients are typically studied for a few isolated in vitro activities. The purpose of this study was to carry out a comprehensive evaluation of the activity on the skin of an association of ingredients from marine and maritime origins using label-free quantitative proteomic analysis, in order to predict the clinical benefits if used in a skin care product. An aqueous gel containing 6.1% of ingredients from marine and maritime origins (amino acid-enriched giant kelp extract, trace element-enriched seawater, dedifferentiated sea fennel cells) was topically applied on human skin explants. The skin explants' proteome was analyzed in a label-free manner by high-performance liquid nano-chromatography coupled with tandem mass spectrometry. A specific data processing pipeline (CORAVALID) providing an objective and comprehensive interpretation of the statistically relevant biological activities processed the results. Compared to untreated skin explants, 64 proteins were significantly regulated by the gel treatment (q-value ≤ 0.05). Computer data processing revealed an activity of the ingredients on the epidermis and the dermis. These significantly regulated proteins are involved in gene expression, cell survival and metabolism, inflammatory processes, dermal extracellular matrix synthesis, melanogenesis and keratinocyte proliferation, migration, and differentiation. These results suggest that the tested ingredients could help to preserve a healthy epidermis and dermis, and possibly to prevent the visible signs of skin aging. © 2018 The Authors. Journal of Cosmetic Dermatology Published by Wiley Periodicals, Inc.

An Automated High Throughput Proteolysis and Desalting Platform for Quantitative Proteomic Analysis

Directory of Open Access Journals (Sweden)

Albert-Baskar Arul

2013-06-01

Full Text Available Proteomics for biomarker validation needs high throughput instrumentation to analyze huge set of clinical samples for quantitative and reproducible analysis at a minimum time without manual experimental errors. Sample preparation, a vital step in proteomics plays a major role in identification and quantification of proteins from biological samples. Tryptic digestion a major check point in sample preparation for mass spectrometry based proteomics needs to be more accurate with rapid processing time. The present study focuses on establishing a high throughput automated online system for proteolytic digestion and desalting of proteins from biological samples quantitatively and qualitatively in a reproducible manner. The present study compares online protein digestion and desalting of BSA with conventional off-line (in-solution method and validated for real time sample for reproducibility. Proteins were identified using SEQUEST data base search engine and the data were quantified using IDEALQ software. The present study shows that the online system capable of handling high throughput samples in 96 well formats carries out protein digestion and peptide desalting efficiently in a reproducible and quantitative manner. Label free quantification showed clear increase of peptide quantities with increase in concentration with much linearity compared to off line method. Hence we would like to suggest that inclusion of this online system in proteomic pipeline will be effective in quantification of proteins in comparative proteomics were the quantification is really very crucial.

Targeted proteomics guided by label-free global proteome analysis in saliva reveal transition signatures from health to periodontal disease.

Science.gov (United States)

Bostanci, Nagihan; Selevsek, Nathalie; Wolski, Witold; Grossmann, Jonas; Bao, Kai; Wahlander, Asa; Trachsel, Christian; Schlapbach, Ralph; Özturk, Veli Özgen; Afacan, Beral; Emingil, Gulnur; Belibasakis, Georgios N

2018-04-02

Periodontal diseases are among the most prevalent worldwide, but largely silent, chronic diseases. They affect the tooth-supporting tissues with multiple ramifications on life quality. Their early diagnosis is still challenging, due to lack of appropriate molecular diagnostic methods. Saliva offers a non-invasively collectable reservoir of clinically relevant biomarkers, which, if utilized efficiently, could facilitate early diagnosis and monitoring of ongoing disease. Despite several novel protein markers being recently enlisted by discovery proteomics, their routine diagnostic application is hampered by the lack of validation platforms that allow for rapid, accurate and simultaneous quantification of multiple proteins in large cohorts. We carried out a pipeline of two proteomic platforms; firstly, we applied open ended label-free quantitative (LFQ) proteomics for discovery in saliva (n=67, health, gingivitis, and periodontitis), followed by selected-reaction monitoring (SRM)-targeted proteomics for validation in an independent cohort (n=82). The LFQ platform led to the discovery of 119 proteins with at least two-fold significant difference between health and disease. The 65 proteins chosen for the subsequent SRM platform included 50 related proteins derived from the significantly enriched processes of the LFQ data, 11 from literature-mining, and four house-keeping ones. Among those, 60 were reproducibly quantifiable proteins (92% success rate), represented by a total of 143 peptides. Machine-learning modeling led to a narrowed-down panel of five proteins of high predictive value for periodontal diseases (higher in disease: Matrix metalloproteinase-9, Ras-related protein-1, Actin-related protein 2/3 complex subunit 5; lower in disease: Clusterin, Deleted in Malignant Brain Tumors 1), with maximum area under the receiver operating curve >0.97. This panel enriches the pool of credible clinical biomarker candidates for diagnostic assay development. Yet, the quantum

Cell Surface Proteome of Dental Pulp Stem Cells Identified by Label-Free Mass Spectrometry.

Directory of Open Access Journals (Sweden)

Christian Niehage

Full Text Available Multipotent mesenchymal stromal cells (MSCs are promising tools for regenerative medicine. They can be isolated from different sources based on their plastic-adherence property. The identification of reliable cell surface markers thus becomes the Holy Grail for their prospective isolation. Here, we determine the cell surface proteomes of human dental pulp-derived MSCs isolated from single donors after culture expansion in low (2% or high (10% serum-containing media. Cell surface proteins were tagged on intact cells using cell impermeable, cleavable sulfo-NHS-SS-biotin, which allows their enrichment by streptavidin pull-down. For the proteomic analyses, we first compared label-free methods to analyze cell surface proteomes i.e. composition, enrichment and proteomic differences, and we developed a new mathematical model to determine cell surface protein enrichment using a combinatorial gene ontology query. Using this workflow, we identified 101 cluster of differentiation (CD markers and 286 non-CD cell surface proteins. Based on this proteome profiling, we identified 14 cell surface proteins, which varied consistently in abundance when cells were cultured under low or high serum conditions. Collectively, our analytical methods provide a basis for identifying the cell surface proteome of dental pulp stem cells isolated from single donors and its evolution during culture or differentiation. Our data provide a comprehensive cell surface proteome for the precise identification of dental pulp-derived MSC populations and their isolation for potential therapeutic intervention.

Label-free proteome profiling reveals developmental-dependent patterns in young barley grains.

Science.gov (United States)

Kaspar-Schoenefeld, Stephanie; Merx, Kathleen; Jozefowicz, Anna Maria; Hartmann, Anja; Seiffert, Udo; Weschke, Winfriede; Matros, Andrea; Mock, Hans-Peter

2016-06-30

Due to its importance as a cereal crop worldwide, high interest in the determination of factors influencing barley grain quality exists. This study focusses on the elucidation of protein networks affecting early grain developmental processes. NanoLC-based separation coupled to label-free MS detection was applied to gain insights into biochemical processes during five different grain developmental phases (pre-storage until storage phase, 3days to 16days after flowering). Multivariate statistics revealed two distinct developmental patterns during the analysed grain developmental phases: proteins showed either highest abundance in the middle phase of development - in the transition phase - or at later developmental stages - within the storage phase. Verification of developmental patterns observed by proteomic analysis was done by applying hypothesis-driven approaches, namely Western Blot analysis and enzyme assays. High general metabolic activity of the grain with regard to protein synthesis, cell cycle regulation, defence against oxidative stress, and energy production via photosynthesis was observed in the transition phase. Proteins upregulated in the storage phase are related towards storage protein accumulation, and interestingly to the defence of storage reserves against pathogens. A mixed regulatory pattern for most enzymes detected in our study points to regulatory mechanisms at the level of protein isoforms. In-depth understanding of early grain developmental processes of cereal caryopses is of high importance as they influence final grain weight and quality. Our knowledge about these processes is still limited, especially on proteome level. To identify key mechanisms in early barley grain development, a label-free data-independent proteomics acquisition approach has been applied. Our data clearly show, that proteins either exhibit highest expression during cellularization and the switch to the storage phase (transition phase, 5-7 DAF), or during storage

Automation of dimethylation after guanidination labeling chemistry and its compatibility with common buffers and surfactants for mass spectrometry-based shotgun quantitative proteome analysis

Energy Technology Data Exchange (ETDEWEB)

Lo, Andy; Tang, Yanan; Chen, Lu; Li, Liang, E-mail: Liang.Li@ualberta.ca

2013-07-25

Graphical abstract: -- Highlights: •Dimethylation after guanidination (2MEGA) uses inexpensive reagents for isotopic labeling of peptides. •2MEGA can be optimized and automated for labeling peptides with high efficiency. •2MEGA is compatible with several commonly used cell lysis and protein solubilization reagents. •The automated 2MEGA labeling method can be used to handle a variety of protein samples for relative proteome quantification. -- Abstract: Isotope labeling liquid chromatography–mass spectrometry (LC–MS) is a major analytical platform for quantitative proteome analysis. Incorporation of isotopes used to distinguish samples plays a critical role in the success of this strategy. In this work, we optimized and automated a chemical derivatization protocol (dimethylation after guanidination, 2MEGA) to increase the labeling reproducibility and reduce human intervention. We also evaluated the reagent compatibility of this protocol to handle biological samples in different types of buffers and surfactants. A commercially available liquid handler was used for reagent dispensation to minimize analyst intervention and at least twenty protein digest samples could be prepared in a single run. Different front-end sample preparation methods for protein solubilization (SDS, urea, Rapigest™, and ProteaseMAX™) and two commercially available cell lysis buffers were evaluated for compatibility with the automated protocol. It was found that better than 94% desired labeling could be obtained in all conditions studied except urea, where the rate was reduced to about 92% due to carbamylation on the peptide amines. This work illustrates the automated 2MEGA labeling process can be used to handle a wide range of protein samples containing various reagents that are often encountered in protein sample preparation for quantitative proteome analysis.

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

Science.gov (United States)

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

2017-08-15

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.

Comparison of one- and two-dimensional liquid chromatography approaches in the label-free quantitative analysis of Methylocella silvestris.

Science.gov (United States)

Patel, Nisha A; Crombie, Andrew; Slade, Susan E; Thalassinos, Konstantinos; Hughes, Chris; Connolly, Joanne B; Langridge, James; Murrell, J Colin; Scrivens, James H

2012-09-07

The proteome of the bacterium Methylocella silvestris has been characterized using reversed phase ultra high pressure liquid chromatography (UPLC) and two-dimensional reversed phase (high pH)-reversed phase (low pH) UPLC prior to mass spectrometric analysis. Variations in protein expression levels were identified with the aid of label-free quantification in a study of soluble protein extracts from the organism grown using methane, succinate, or propane as a substrate. The number of first dimensional fractionation steps has been varied for 2D analyses, and the impact on data throughput and quality has been demonstrated. Comparisons have been made regarding required experimental considerations including total loading of biological samples required, instrument time, and resulting data file sizes. The data obtained have been evaluated with respect to number of protein identifications, confidence of assignments, sequence coverage, relative levels of proteins, and dynamic range. Good qualitative and quantitative agreement was observed between the different approaches, and the potential benefits and limitations of the reversed phase-reversed phase UPLC technique in label-free analysis are discussed. A preliminary screen of the protein regulation data has also been performed, providing evidence for a possible propane assimilation route.

Spatial and molecular resolution of diffuse malignant mesothelioma heterogeneity by integrating label-free FTIR imaging, laser capture microdissection and proteomics

Science.gov (United States)

Großerueschkamp, Frederik; Bracht, Thilo; Diehl, Hanna C.; Kuepper, Claus; Ahrens, Maike; Kallenbach-Thieltges, Angela; Mosig, Axel; Eisenacher, Martin; Marcus, Katrin; Behrens, Thomas; Brüning, Thomas; Theegarten, Dirk; Sitek, Barbara; Gerwert, Klaus

2017-03-01

Diffuse malignant mesothelioma (DMM) is a heterogeneous malignant neoplasia manifesting with three subtypes: epithelioid, sarcomatoid and biphasic. DMM exhibit a high degree of spatial heterogeneity that complicates a thorough understanding of the underlying different molecular processes in each subtype. We present a novel approach to spatially resolve the heterogeneity of a tumour in a label-free manner by integrating FTIR imaging and laser capture microdissection (LCM). Subsequent proteome analysis of the dissected homogenous samples provides in addition molecular resolution. FTIR imaging resolves tumour subtypes within tissue thin-sections in an automated and label-free manner with accuracy of about 85% for DMM subtypes. Even in highly heterogeneous tissue structures, our label-free approach can identify small regions of interest, which can be dissected as homogeneous samples using LCM. Subsequent proteome analysis provides a location specific molecular characterization. Applied to DMM subtypes, we identify 142 differentially expressed proteins, including five protein biomarkers commonly used in DMM immunohistochemistry panels. Thus, FTIR imaging resolves not only morphological alteration within tissue but it resolves even alterations at the level of single proteins in tumour subtypes. Our fully automated workflow FTIR-guided LCM opens new avenues collecting homogeneous samples for precise and predictive biomarkers from omics studies.

Study of early leaf senescence in Arabidopsis thaliana by quantitative proteomics using reciprocal N-14/N-15 Labeling and difference gel electrophoresis

NARCIS (Netherlands)

Hebeler, Romano; Oeljeklaus, Silke; Reidegeld, Kai E.; Eisenacher, Martin; Stephan, Christian; Sitek, Barbara; Stuehler, Kai; Meyer, Helmut E.; Sturre, Marcel J. G.; Dijkwel, Paul P.; Warscheid, Bettina

Leaf senescence represents the final stage of leaf development and is associated with fundamental changes on the level of the proteome. For the quantitative analysis of changes in protein abundance related to early leaf senescence, we designed an elaborate double and reverse labeling strategy

Label-free proteomic analysis of intestinal mucosa proteins in common carp (Cyprinus carpio) infected with Aeromonas hydrophila.

Science.gov (United States)

Di, Guilan; Li, Hui; Zhang, Chao; Zhao, Yanjing; Zhou, Chuanjiang; Naeem, Sajid; Li, Li; Kong, Xianghui

2017-07-01

Outbreaks of infectious diseases in common carp Cyprinus carpio, a major cultured fish in northern regions of China, constantly result in significant economic losses. Until now, information proteomic on immune defence remains limited. In the present study, a profile of intestinal mucosa immune response in Cyprinus carpio was investigated after 0, 12, 36 and 84 h after challenging tissues with Aeromonas hydrophila at a concentration of 1.4 × 10 8  CFU/mL. Proteomic profiles in different samples were compared using label-free quantitative proteomic approach. Based on MASCOT database search, 1149 proteins were identified in samples after normalisation of proteins. Treated groups 1 (T1) and 2 (T2) were first clustered together and then clustered with control (C group). The distance between C and treated group 3 (T3) represented the maxima according to hierarchical cluster analysis. Therefore, comparative analysis between C and T3 was selected in the following analysis. A total of 115 proteins with differential abundance were detected to show conspicuous expressing variances. A total of 52 up-regulated proteins and 63 down-regulated proteins were detected in T3. Gene ontology analysis showed that identified up-regulated differentially expressed proteins in T3 were mainly localised in the hemoglobin complex, and down-regulated proteins in T3 were mainly localised in the major histocompatibility complex II protein complex. Forty-six proteins of differential abundance (40% of 115) were involved in immune response, with 17 up-regulated and 29 down-regulated proteins detected in T3. This study is the first to report proteome response of carp intestinal mucosa against A. hydrophila infection; information obtained contribute to understanding defence mechanisms of carp intestinal mucosa. Copyright © 2017 Elsevier Ltd. All rights reserved.

Completion of proteomic data sets by Kd measurement using cell-free synthesis of site-specifically labeled proteins.

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Paul Majkut

Full Text Available The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values. In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2's were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level.

Label-Free Proteomic Identification of Endogenous, Insulin-Stimulated Interaction Partners of Insulin Receptor Substrate-1

Science.gov (United States)

Geetha, Thangiah; Langlais, Paul; Luo, Moulun; Mapes, Rebekka; Lefort, Natalie; Chen, Shu-Chuan; Mandarino, Lawrence J.; Yi, Zhengping

2011-03-01

Protein-protein interactions are key to most cellular processes. Tandem mass spectrometry (MS/MS)-based proteomics combined with co-immunoprecipitation (CO-IP) has emerged as a powerful approach for studying protein complexes. However, a majority of systematic proteomics studies on protein-protein interactions involve the use of protein overexpression and/or epitope-tagged bait proteins, which might affect binding stoichiometry and lead to higher false positives. Here, we report an application of a straightforward, label-free CO-IP-MS/MS method, without the use of protein overexpression or protein tags, to the investigation of changes in the abundance of endogenous proteins associated with a bait protein, which is in this case insulin receptor substrate-1 (IRS-1), under basal and insulin stimulated conditions. IRS-1 plays a central role in the insulin signaling cascade. Defects in the protein-protein interactions involving IRS-1 may lead to the development of insulin resistance and type 2 diabetes. HPLC-ESI-MS/MS analyses identified eleven novel endogenous insulin-stimulated IRS-1 interaction partners in L6 myotubes reproducibly, including proteins play an important role in protein dephosphorylation [protein phosphatase 1 regulatory subunit 12A, (PPP1R12A)], muscle contraction and actin cytoskeleton rearrangement, endoplasmic reticulum stress, and protein folding, as well as protein synthesis. This novel application of label-free CO-IP-MS/MS quantification to assess endogenous interaction partners of a specific protein will prove useful for understanding how various cell stimuli regulate insulin signal transduction.

Analysis of high accuracy, quantitative proteomics data in the MaxQB database.

Science.gov (United States)

Schaab, Christoph; Geiger, Tamar; Stoehr, Gabriele; Cox, Juergen; Mann, Matthias

2012-03-01

MS-based proteomics generates rapidly increasing amounts of precise and quantitative information. Analysis of individual proteomic experiments has made great strides, but the crucial ability to compare and store information across different proteome measurements still presents many challenges. For example, it has been difficult to avoid contamination of databases with low quality peptide identifications, to control for the inflation in false positive identifications when combining data sets, and to integrate quantitative data. Although, for example, the contamination with low quality identifications has been addressed by joint analysis of deposited raw data in some public repositories, we reasoned that there should be a role for a database specifically designed for high resolution and quantitative data. Here we describe a novel database termed MaxQB that stores and displays collections of large proteomics projects and allows joint analysis and comparison. We demonstrate the analysis tools of MaxQB using proteome data of 11 different human cell lines and 28 mouse tissues. The database-wide false discovery rate is controlled by adjusting the project specific cutoff scores for the combined data sets. The 11 cell line proteomes together identify proteins expressed from more than half of all human genes. For each protein of interest, expression levels estimated by label-free quantification can be visualized across the cell lines. Similarly, the expression rank order and estimated amount of each protein within each proteome are plotted. We used MaxQB to calculate the signal reproducibility of the detected peptides for the same proteins across different proteomes. Spearman rank correlation between peptide intensity and detection probability of identified proteins was greater than 0.8 for 64% of the proteome, whereas a minority of proteins have negative correlation. This information can be used to pinpoint false protein identifications, independently of peptide database

Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

Science.gov (United States)

Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

2010-05-01

Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

Magnetoresistive biosensors for quantitative proteomics

Science.gov (United States)

Zhou, Xiahan; Huang, Chih-Cheng; Hall, Drew A.

2017-08-01

Quantitative proteomics, as a developing method for study of proteins and identification of diseases, reveals more comprehensive and accurate information of an organism than traditional genomics. A variety of platforms, such as mass spectrometry, optical sensors, electrochemical sensors, magnetic sensors, etc., have been developed for detecting proteins quantitatively. The sandwich immunoassay is widely used as a labeled detection method due to its high specificity and flexibility allowing multiple different types of labels. While optical sensors use enzyme and fluorophore labels to detect proteins with high sensitivity, they often suffer from high background signal and challenges in miniaturization. Magnetic biosensors, including nuclear magnetic resonance sensors, oscillator-based sensors, Hall-effect sensors, and magnetoresistive sensors, use the specific binding events between magnetic nanoparticles (MNPs) and target proteins to measure the analyte concentration. Compared with other biosensing techniques, magnetic sensors take advantage of the intrinsic lack of magnetic signatures in biological samples to achieve high sensitivity and high specificity, and are compatible with semiconductor-based fabrication process to have low-cost and small-size for point-of-care (POC) applications. Although still in the development stage, magnetic biosensing is a promising technique for in-home testing and portable disease monitoring.

Label-Free Quantitative Analysis of Mitochondrial Proteomes Using the Multienzyme Digestion-Filter Aided Sample Preparation (MED-FASP) and "Total Protein Approach".

Science.gov (United States)

Wiśniewski, Jacek R

2017-01-01

Determination of proteome composition and measuring of changes in protein titers provide important information with a substantial value for studying mitochondria.This chapter describes a workflow for the quantitative analysis of mitochondrial proteome with a focus on sample preparation and quantitative analysis of the data. The workflow involves the multienzyme digestion-filter aided sample preparation (MED-FASP) protocol enabling efficient extraction of proteins and high rate of protein-to-peptide conversion. Consecutive protein digestion with Lys C and trypsin enables generation of peptide fractions with minimal overlap, largely increases the number of identified proteins, and extends their sequence coverage. Abundances of proteins identified by multiple peptides can be assessed by the "Total Protein Approach."

Extracting histones for the specific purpose of label-free MS.

Science.gov (United States)

Govaert, Elisabeth; Van Steendam, Katleen; Scheerlinck, Ellen; Vossaert, Liesbeth; Meert, Paulien; Stella, Martina; Willems, Sander; De Clerck, Laura; Dhaenens, Maarten; Deforce, Dieter

2016-12-01

Extracting histones from cells is the first step in studies that aim to characterize histones and their post-translational modifications (hPTMs) with MS. In the last decade, label-free quantification is more frequently being used for MS-based histone characterization. However, many histone extraction protocols were not specifically designed for label-free MS. While label-free quantification has its advantages, it is also very susceptible to technical variation. Here, we adjust an established histone extraction protocol according to general label-free MS guidelines with a specific focus on minimizing sample handling. These protocols are first evaluated using SDS-PAGE. Hereafter, a selection of extraction protocols was used in a complete histone workflow for label-free MS. All protocols display nearly identical relative quantification of hPTMs. We thus show that, depending on the cell type under investigation and at the cost of some additional contaminating proteins, minimizing sample handling can be done during histone isolation. This allows analyzing bigger sample batches, leads to reduced technical variation and minimizes the chance of in vitro alterations to the hPTM snapshot. Overall, these results allow researchers to determine the best protocol depending on the resources and goal of their specific study. Data are available via ProteomeXchange with identifier PXD002885. © 2016 The Authors. Proteomics Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Mapping Protein-Protein Interactions by Quantitative Proteomics

DEFF Research Database (Denmark)

Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

2010-01-01

spectrometry (MS)-based proteomics in combination with affinity purification protocols has become the method of choice to map and track the dynamic changes in protein-protein interactions, including the ones occurring during cellular signaling events. Different quantitative MS strategies have been used...... to characterize protein interaction networks. In this chapter we describe in detail the use of stable isotope labeling by amino acids in cell culture (SILAC) for the quantitative analysis of stimulus-dependent dynamic protein interactions.......Proteins exert their function inside a cell generally in multiprotein complexes. These complexes are highly dynamic structures changing their composition over time and cell state. The same protein may thereby fulfill different functions depending on its binding partners. Quantitative mass...

Data in support of quantitative proteomics to identify potential virulence regulators in Paracoccidioides brasiliensis isolates

Directory of Open Access Journals (Sweden)

Alexandre Keiji Tashima

2015-12-01

Full Text Available Paracoccidioides genus are the etiologic agents of paracoccidioidomycosis (PCM, a systemic mycosis endemic in Latin America. Few virulence factors have been identified in these fungi. This paper describes support data from the quantitative proteomics of Paracoccidioides brasiliensis attenuated and virulent isolates [1]. The protein compositions of two isolates of the Pb18 strain showing distinct infection profiles were quantitatively assessed by stable isotopic dimethyl labeling and proteomic analysis. The mass spectrometry and the analysis dataset have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with identifier PXD000804.

Benchmarking sample preparation/digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics.

Science.gov (United States)

Muller, Leslie; Fornecker, Luc; Van Dorsselaer, Alain; Cianférani, Sarah; Carapito, Christine

2016-12-01

Sample preparation, typically by in-solution or in-gel approaches, has a strong influence on the accuracy and robustness of quantitative proteomics workflows. The major benefit of in-gel procedures is their compatibility with detergents (such as SDS) for protein solubilization. However, SDS-PAGE is a time-consuming approach. Tube-gel (TG) preparation circumvents this drawback as it involves directly trapping the sample in a polyacrylamide gel matrix without electrophoresis. We report here the first global label-free quantitative comparison between TG, stacking gel (SG), and basic liquid digestion (LD). A series of UPS1 standard mixtures (at 0.5, 1, 2.5, 5, 10, and 25 fmol) were spiked in a complex yeast lysate background. TG preparation allowed more yeast proteins to be identified than did the SG and LD approaches, with mean numbers of 1979, 1788, and 1323 proteins identified, respectively. Furthermore, the TG method proved equivalent to SG and superior to LD in terms of the repeatability of the subsequent experiments, with mean CV for yeast protein label-free quantifications of 7, 9, and 10%. Finally, known variant UPS1 proteins were successfully detected in the TG-prepared sample within a complex background with high sensitivity. All the data from this study are accessible on ProteomeXchange (PXD003841). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Condenser: a statistical aggregation tool for multi-sample quantitative proteomic data from Matrix Science Mascot Distiller™.

Science.gov (United States)

Knudsen, Anders Dahl; Bennike, Tue; Kjeldal, Henrik; Birkelund, Svend; Otzen, Daniel Erik; Stensballe, Allan

2014-05-30

We describe Condenser, a freely available, comprehensive open-source tool for merging multidimensional quantitative proteomics data from the Matrix Science Mascot Distiller Quantitation Toolbox into a common format ready for subsequent bioinformatic analysis. A number of different relative quantitation technologies, such as metabolic (15)N and amino acid stable isotope incorporation, label-free and chemical-label quantitation are supported. The program features multiple options for curative filtering of the quantified peptides, allowing the user to choose data quality thresholds appropriate for the current dataset, and ensure the quality of the calculated relative protein abundances. Condenser also features optional global normalization, peptide outlier removal, multiple testing and calculation of t-test statistics for highlighting and evaluating proteins with significantly altered relative protein abundances. Condenser provides an attractive addition to the gold-standard quantitative workflow of Mascot Distiller, allowing easy handling of larger multi-dimensional experiments. Source code, binaries, test data set and documentation are available at http://condenser.googlecode.com/. Copyright © 2014 Elsevier B.V. All rights reserved.

Data set for the proteomic inventory and quantitative analysis of chicken uterine fluid during eggshell biomineralization

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Pauline Marie

2014-12-01

Full Text Available Chicken eggshell is the protective barrier of the egg. It is a biomineral composed of 95% calcium carbonate on calcitic form and 3.5% organic matrix proteins. Mineralization process occurs in uterus into the uterine fluid. This acellular fluid contains ions and organic matrix proteins precursors which are interacting with the mineral phase and control crystal growth, eggshell structure and mechanical properties. We performed a proteomic approach and identified 308 uterine fluid proteins. Gene Ontology terms enrichments were determined to investigate their potential functions. Mass spectrometry analyses were also combined to label free quantitative analysis to determine the relative abundance of 96 proteins at initiation, rapid growth phase and termination of shell calcification. Sixty four showed differential abundance according to the mineralization stage. Their potential functions have been annotated. The complete proteomic, bioinformatic and functional analyses are reported in Marie et al., J. Proteomics (2015 [1].

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

International Nuclear Information System (INIS)

Yu, Qing; Shi, Xudong; Feng, Yu; Kent, K. Craig; Li, Lingjun

2017-01-01

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

Improving data quality and preserving HCD-generated reporter ions with EThcD for isobaric tag-based quantitative proteomics and proteome-wide PTM studies

Energy Technology Data Exchange (ETDEWEB)

Yu, Qing [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Shi, Xudong [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Feng, Yu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Kent, K. Craig [Department of Surgery, School of Medicine and Public Health, University of Wisconsin, Madison, WI 53705 (United States); Li, Lingjun, E-mail: lingjun.li@wisc.edu [School of Pharmacy, University of Wisconsin, Madison, WI 53705 (United States); Department of Chemistry, University of Wisconsin, Madison, WI 53706 (United States)

2017-05-22

Mass spectrometry (MS)-based isobaric labeling has undergone rapid development in recent years due to its capability for high throughput quantitation. Apart from its originally designed use with collision-induced dissociation (CID) and higher-energy collisional dissociation (HCD), isobaric tagging technique could also work with electron-transfer dissociation (ETD), which provides complementarity to CID and is preferred in sequencing peptides with post-translational modifications (PTMs). However, ETD suffers from long reaction time, reduced duty cycle and bias against peptides with lower charge states. In addition, common fragmentation mechanism in ETD results in altered reporter ion production, decreased multiplexing capability, and even loss of quantitation capability for some of the isobaric tags, including custom-designed dimethyl leucine (DiLeu) tags. Here, we demonstrate a novel electron-transfer/higher-energy collision dissociation (EThcD) approach that preserves original reporter ion channels, mitigates bias against lower charge states, improves sensitivity, and significantly improves data quality for quantitative proteomics and proteome-wide PTM studies. Systematic optimization was performed to achieve a balance between data quality and sensitivity. We provide direct comparison of EThcD with ETD and HCD for DiLeu- and TMT-labeled HEK cell lysate and IMAC enriched phosphopeptides. Results demonstrate improved data quality and phosphorylation localization accuracy while preserving sufficient reporter ion production. Biological studies were performed to investigate phosphorylation changes in a mouse vascular smooth muscle cell line treated with four different conditions. Overall, EThcD exhibits superior performance compared to conventional ETD and offers distinct advantages compared to HCD in isobaric labeling based quantitative proteomics and quantitative PTM studies. - Highlights: • EThcD was optimized for isobaric tag-labeled peptides for quantitative

Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry.

Science.gov (United States)

Scheerlinck, E; Dhaenens, M; Van Soom, A; Peelman, L; De Sutter, P; Van Steendam, K; Deforce, D

2015-12-01

Sample preparation is the crucial starting point to obtain high-quality mass spectrometry data and can be divided into two main steps in a bottom-up proteomics approach: cell/tissue lysis with or without detergents and a(n) (in-solution) digest comprising denaturation, reduction, alkylation, and digesting of the proteins. Here, some important considerations, among others, are that the reagents used for sample preparation can inhibit the digestion enzyme (e.g., 0.1% sodium dodecyl sulfate [SDS] and 0.5 M guanidine HCl), give rise to ion suppression (e.g., polyethylene glycol [PEG]), be incompatible with liquid chromatography-tandem mass spectrometry (LC-MS/MS) (e.g., SDS), and can induce additional modifications (e.g., urea). Taken together, all of these irreproducible effects are gradually becoming a problem when label-free quantitation of the samples is envisioned such as during the increasingly popular high-definition mass spectrometry (HDMS(E)) and sequential window acquisition of all theoretical fragment ion spectra (SWATH) data-independent acquisition strategies. Here, we describe the detailed validation of a reproducible method with sufficient protein yield for sample preparation without any known LC-MS/MS interfering substances by using 1% sodium deoxycholate (SDC) during both cell lysis and in-solution digest. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

A qualitative and quantitative evaluation of the peptide characteristics of microwave- and ultrasound-assisted digestion in discovery and targeted proteomic analyses.

Science.gov (United States)

Guo, Zhengguang; Cheng, Jie; Sun, Haidan; Sun, Wei

2017-08-30

Fast digestion methods can dramatically accelerate enzyme digestion and increase the throughput of proteomic analysis. However, the peptide characteristics of fast digestion methods and their performance in discovery and targeted proteomic analysis must be systematically evaluated. Three digestion methods, including overnight digestion, microwave-assisted protein enzymatic digestion (MAPED), and high-intensity focused ultrasonic-assisted enzymatic digestion (HIFUSAED), in trypsin or in trypsin/Lys-C were comprehensively compared in both discovery and targeted proteomics analysis using the HeLa cell proteome. In discovery proteomic analysis, the highest numbers of peptides and proteins were identified when the sample was digested via the MAPED method with trypsin/Lys-C. The fast digestion methods showed a higher mis-cleavage rate and a lower semi-tryptic rate than the overnight digestion method. In both label-free quantitative analysis and targeted proteomic analysis, both fully cleaved peptides (FCPs) and mis-cleaved peptides (MCPs) from the fast digestion methods and the overnight digestion method showed good reproducibility if they showed good abundance. When both the FCPs and MCPs were included in the analysis, the MAPED with trypsin/Lys-C method showed the best results for both discovery proteomic analysis and relative quantitative targeted proteomic analysis. These results will be beneficial for the application of fast digestion methods to proteomics. Copyright © 2017 John Wiley & Sons, Ltd.

In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

Directory of Open Access Journals (Sweden)

Esther N Pesciotta

Full Text Available Diamond Blackfan Anemia (DBA is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform

Science.gov (United States)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-11-01

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (r

Quantitative Proteomic Analysis of Mouse Embryonic Fibroblasts and Induced Pluripotent Stem Cells Using 16O /18O labeling

Energy Technology Data Exchange (ETDEWEB)

Huang, Xin; Tian, Changhai; Liu, Miao; Wang, Yongxiang; Tolmachev, Aleksey V.; Sharma, Seema; Yu, Fang; Fu, Kai; Zheng, Jialin; Ding, Shi-Jian

2012-04-06

Induced pluripotent stem cells (iPSC) hold great promise for regenerative medicine as well as for investigations into the pathogenesis and treatment of various diseases. Understanding of key intracellular signaling pathways and protein targets that control development of iPSC from somatic cells is essential for designing new approaches to improve reprogramming efficiency. Here we report the development and application of an integrated quantitative proteomics platform for investigating differences in protein expressions between mouse embryonic fibroblasts (MEF) and MEF-derived iPSC. This platform consists of 16O/18O labeling, multidimensional peptide separation coupled with tandem mass spectrometry, and data analysis with UNiquant software. Using this platform a total of 2,481 proteins were identified and quantified from the 16O/18O-labeled MEF-iPSC proteome mixtures with a false discovery rate of 0.01. Among them, 218 proteins were significantly upregulated, while 247 proteins were significantly downregulated in iPSC compared to MEF. Many nuclear proteins, including Hdac1, Dnmt1, Pcna, Ccnd1, Smarcc1, and subunits in DNA replication and RNA polymerase II complex were found to be enhanced in iPSC. Protein network analysis revealed that Pcna functions as a hub orchestrating complicated mechanisms including DNA replication, epigenetic inheritance (Dnmt1) and chromatin remodeling (Smarcc1) to reprogram MEF and maintain stemness of iPSC.

Establishment of Dimethyl Labeling-based Quantitative Acetylproteomics in Arabidopsis.

Science.gov (United States)

Liu, Shichang; Yu, Fengchao; Yang, Zhu; Wang, Tingliang; Xiong, Hairong; Chang, Caren; Yu, Weichuan; Li, Ning

2018-05-01

Protein acetylation, one of many types of post-translational modifications (PTMs), is involved in a variety of biological and cellular processes. In the present study, we applied both C sCl d ensity g radient (CDG) centrifugation-based protein fractionation and a dimethyl-labeling-based 4C quantitative PTM proteomics workflow in the study of dynamic acetylproteomic changes in Arabidopsis. This workflow integrates the dimethyl c hemical labeling with c hromatography-based acetylpeptide separation and enrichment followed by mass spectrometry (MS) analysis, the extracted ion chromatogram (XIC) quantitation-based c omputational analysis of mass spectrometry data to measure dynamic changes of acetylpeptide level using an in-house software program, named S table isotope-based Qua ntitation- D imethyl labeling (SQUA-D), and finally the c onfirmation of ethylene hormone-regulated acetylation using immunoblot analysis. Eventually, using this proteomic approach, 7456 unambiguous acetylation sites were found from 2638 different acetylproteins, and 5250 acetylation sites, including 5233 sites on lysine side chain and 17 sites on protein N termini, were identified repetitively. Out of these repetitively discovered acetylation sites, 4228 sites on lysine side chain ( i.e. 80.5%) are novel. These acetylproteins are exemplified by the histone superfamily, ribosomal and heat shock proteins, and proteins related to stress/stimulus responses and energy metabolism. The novel acetylproteins enriched by the CDG centrifugation fractionation contain many cellular trafficking proteins, membrane-bound receptors, and receptor-like kinases, which are mostly involved in brassinosteroid, light, gravity, and development signaling. In addition, we identified 12 highly conserved acetylation site motifs within histones, P-glycoproteins, actin depolymerizing factors, ATPases, transcription factors, and receptor-like kinases. Using SQUA-D software, we have quantified 33 ethylene hormone-enhanced and

On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics

Directory of Open Access Journals (Sweden)

Benedetta Turriziani

2014-04-01

Full Text Available With the advent of the “-omics” era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.

Gel-free/label-free proteomic analysis of root tip of soybean over time under flooding and drought stresses.

Science.gov (United States)

Wang, Xin; Oh, MyeongWon; Sakata, Katsumi; Komatsu, Setsuko

2016-01-01

Growth in the early stage of soybean is markedly inhibited under flooding and drought stresses. To explore the responsive mechanisms of soybean, temporal protein profiles of root tip under flooding and drought stresses were analyzed using gel-free/label-free proteomic technique. Root tip was analyzed because it was the most sensitive organ against flooding, and it was beneficial to root penetration under drought. UDP glucose: glycoprotein glucosyltransferase was decreased and increased in soybean root under flooding and drought, respectively. Temporal protein profiles indicated that fermentation and protein synthesis/degradation were essential in root tip under flooding and drought, respectively. In silico protein-protein interaction analysis revealed that the inductive and suppressive interactions between S-adenosylmethionine synthetase family protein and B-S glucosidase 44 under flooding and drought, respectively, which are related to carbohydrate metabolism. Furthermore, biotin/lipoyl attachment domain containing protein and Class II aminoacyl tRNA/biotin synthetases superfamily protein were repressed in the root tip during time-course stresses. These results suggest that biotin and biotinylation might be involved in energy management to cope with flooding and drought in early stage of soybean-root tip. Copyright © 2015 Elsevier B.V. All rights reserved.

Proteomics wants cRacker: automated standardized data analysis of LC-MS derived proteomic data.

Science.gov (United States)

Zauber, Henrik; Schulze, Waltraud X

2012-11-02

The large-scale analysis of thousands of proteins under various experimental conditions or in mutant lines has gained more and more importance in hypothesis-driven scientific research and systems biology in the past years. Quantitative analysis by large scale proteomics using modern mass spectrometry usually results in long lists of peptide ion intensities. The main interest for most researchers, however, is to draw conclusions on the protein level. Postprocessing and combining peptide intensities of a proteomic data set requires expert knowledge, and the often repetitive and standardized manual calculations can be time-consuming. The analysis of complex samples can result in very large data sets (lists with several 1000s to 100,000 entries of different peptides) that cannot easily be analyzed using standard spreadsheet programs. To improve speed and consistency of the data analysis of LC-MS derived proteomic data, we developed cRacker. cRacker is an R-based program for automated downstream proteomic data analysis including data normalization strategies for metabolic labeling and label free quantitation. In addition, cRacker includes basic statistical analysis, such as clustering of data, or ANOVA and t tests for comparison between treatments. Results are presented in editable graphic formats and in list files.

Single Cell Immuno-Laser Microdissection Coupled to Label-Free Proteomics to Reveal the Proteotypes of Human Brain Cells After Ischemia.

Science.gov (United States)

García-Berrocoso, Teresa; Llombart, Víctor; Colàs-Campàs, Laura; Hainard, Alexandre; Licker, Virginie; Penalba, Anna; Ramiro, Laura; Simats, Alba; Bustamante, Alejandro; Martínez-Saez, Elena; Canals, Francesc; Sanchez, Jean-Charles; Montaner, Joan

2018-01-01

Cerebral ischemia entails rapid tissue damage in the affected brain area causing devastating neurological dysfunction. How each component of the neurovascular unit contributes or responds to the ischemic insult in the context of the human brain has not been solved yet. Thus, the analysis of the proteome is a straightforward approach to unraveling these cell proteotypes. In this study, post-mortem brain slices from ischemic stroke patients were obtained corresponding to infarcted (IC) and contralateral (CL) areas. By means of laser microdissection, neurons and blood brain barrier structures (BBB) were isolated and analyzed using label-free quantification. MS data are available via ProteomeXchange with identifier PXD003519. Ninety proteins were identified only in neurons, 260 proteins only in the BBB and 261 proteins in both cell types. Bioinformatics analyses revealed that repair processes, mainly related to synaptic plasticity, are outlined in microdissected neurons, with nonexclusive important functions found in the BBB. A total of 30 proteins showing p 2 between IC and CL areas were considered meaningful in this study: 13 in neurons, 14 in the BBB and 3 in both cell types. Twelve of these proteins were selected as candidates and analyzed by immunohistofluorescence in independent brains. The MS findings were completely verified for neuronal SAHH2 and SRSF1 whereas the presence in both cell types of GABT and EAA2 was only validated in neurons. In addition, SAHH2 showed its potential as a prognostic biomarker of neurological improvement when analyzed early in the plasma of ischemic stroke patients. Therefore, the quantitative proteomes of neurons and the BBB (or proteotypes) after human brain ischemia presented here contribute to increasing the knowledge regarding the molecular mechanisms of ischemic stroke pathology and highlight new proteins that might represent putative biomarkers of brain ischemia or therapeutic targets. © 2018 by The American Society for

Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

Science.gov (United States)

Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

2017-04-01

The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

High-performance hybrid Orbitrap mass spectrometers for quantitative proteome analysis

DEFF Research Database (Denmark)

Williamson, James C; Edwards, Alistair V G; Verano-Braga, Thiago

2016-01-01

We present basic workups and quantitative comparisons for two current generation Orbitrap mass spectrometers, the Q Exactive Plus and Orbitrap Fusion Tribrid, which are widely considered two of the highest performing instruments on the market. We assessed the performance of two quantitative methods...... on both instruments, namely label-free quantitation and stable isotope labeling using isobaric tags, for studying the heat shock response in Escherichia coli. We investigated the recently reported MS3 method on the Fusion instrument and the potential of MS3-based reporter ion isolation Synchronous...... Precursor Selection (SPS) and its impact on quantitative accuracy. We confirm that the label-free approach offers a more linear response with a wider dynamic range than MS/MS-based isobaric tag quantitation and that the MS3/SPS approach alleviates but does not eliminate dynamic range compression. We...

Quantitative proteomic analysis of microdissected oral epithelium for cancer biomarker discovery.

Science.gov (United States)

Xiao, Hua; Langerman, Alexander; Zhang, Yan; Khalid, Omar; Hu, Shen; Cao, Cheng-Xi; Lingen, Mark W; Wong, David T W

2015-11-01

Specific biomarkers are urgently needed for the detection and progression of oral cancer. The objective of this study was to discover cancer biomarkers from oral epithelium through utilizing high throughput quantitative proteomics approaches. Morphologically malignant, epithelial dysplasia, and adjacent normal epithelial tissues were laser capture microdissected (LCM) from 19 patients and used for proteomics analysis. Total proteins from each group were extracted, digested and then labelled with corresponding isobaric tags for relative and absolute quantitation (iTRAQ). Labelled peptides from each sample were combined and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) for protein identification and quantification. In total, 500 proteins were identified and 425 of them were quantified. When compared with adjacent normal oral epithelium, 17 and 15 proteins were consistently up-regulated or down-regulated in malignant and epithelial dysplasia, respectively. Half of these candidate biomarkers were discovered for oral cancer for the first time. Cornulin was initially confirmed in tissue protein extracts and was further validated in tissue microarray. Its presence in the saliva of oral cancer patients was also explored. Myoglobin and S100A8 were pre-validated by tissue microarray. These data demonstrated that the proteomic biomarkers discovered through this strategy are potential targets for oral cancer detection and salivary diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Real-time label-free quantitative fluorescence microscopy-based detection of ATP using a tunable fluorescent nano-aptasensor platform.

Science.gov (United States)

Shrivastava, Sajal; Sohn, Il-Yung; Son, Young-Min; Lee, Won-Il; Lee, Nae-Eung

2015-12-14

Although real-time label-free fluorescent aptasensors based on nanomaterials are increasingly recognized as a useful strategy for the detection of target biomolecules with high fidelity, the lack of an imaging-based quantitative measurement platform limits their implementation with biological samples. Here we introduce an ensemble strategy for a real-time label-free fluorescent graphene (Gr) aptasensor platform. This platform employs aptamer length-dependent tunability, thus enabling the reagentless quantitative detection of biomolecules through computational processing coupled with real-time fluorescence imaging data. We demonstrate that this strategy effectively delivers dose-dependent quantitative readouts of adenosine triphosphate (ATP) concentration on chemical vapor deposited (CVD) Gr and reduced graphene oxide (rGO) surfaces, thereby providing cytotoxicity assessment. Compared with conventional fluorescence spectrometry methods, our highly efficient, universally applicable, and rational approach will facilitate broader implementation of imaging-based biosensing platforms for the quantitative evaluation of a range of target molecules.

Label-free Proteomic Reveals that Cowpea Severe Mosaic Virus Transiently Suppresses the Host Leaf Protein Accumulation During the Compatible Interaction with Cowpea (Vigna unguiculata [L.] Walp.).

Science.gov (United States)

Paiva, Ana L S; Oliveira, Jose T A; de Souza, Gustavo A; Vasconcelos, Ilka M

2016-12-02

Viruses are important plant pathogens that threaten diverse crops worldwide. Diseases caused by Cowpea severe mosaic virus (CPSMV) have drawn attention because of the serious damages they cause to economically important crops including cowpea. This work was undertaken to quantify and identify the responsive proteins of a susceptible cowpea genotype infected with CPSMV, in comparison with mock-inoculated controls, using label-free quantitative proteomics and databanks, aiming at providing insights on the molecular basis of this compatible interaction. Cowpea leaves were mock- or CPSMV-inoculated and 2 and 6 days later proteins were extracted and analyzed. More than 3000 proteins were identified (data available via ProteomeXchange, identifier PXD005025) and 75 and 55 of them differentially accumulated in response to CPSMV, at 2 and 6 DAI, respectively. At 2 DAI, 76% of the proteins decreased in amount and 24% increased. However, at 6 DAI, 100% of the identified proteins increased. Thus, CPSMV transiently suppresses the synthesis of proteins involved particularly in the redox homeostasis, protein synthesis, defense, stress, RNA/DNA metabolism, signaling, and other functions, allowing viral invasion and spread in cowpea tissues.

Dissecting the C. elegans response during infection using quantitative proteomics

DEFF Research Database (Denmark)

Simonsen, Karina Trankjær; Møller-Jensen, Jakob; Kristensen, Anders Riis

2008-01-01

The adherent invasive E. coli isolated from patients with Crohn’s disease in humans is pathogenic for C. elegans. We show here that when C. elegans feeds on the pathogenic E. coli, the life span is shortened significantly compared to the normal laboratory food, the OP50 E. coli. In this study...... the infection process is followed using GFP-expressing bacteria and persistence assays. A quantitative proteomic approach was used to follow the C. elegans host response during the infection process. C. elegans were metabolic labeled with the stable isotope 15N and samples from three different time points......, many of which also have been found in studies using other pathogens. So far, large-scale investigations of the C. elegans immune response have been performed using micro-arrays. This study is the first to make use of quantitative proteomics to directly follow the protein dynamics during the infection...

Characterization of global yeast quantitative proteome data generated from the wild-type and glucose repression Saccharomyces cerevisiae strains: The comparison of two quantitative methods

DEFF Research Database (Denmark)

Usaite, Renata; Wohlschlegel, James; Venable, John D.

2008-01-01

The quantitative proteomic analysis of complex protein mixtures is emerging as a technically challenging but viable systems-level approach for studying cellular function. This study presents a large-scale comparative analysis of protein abundances from yeast protein lysates derived from both wild......-type yeast and yeast strains lacking key components of the Snf1 kinase complex. Four different strains were grown under well-controlled chemostat conditions. Multidimensional protein identification technology followed by quantitation using either spectral counting or stable isotope labeling approaches...... labeling strategy. The stable isotope labeling based quantitative approach was found to be highly reproducible among biological replicates when complex protein mixtures containing small expression changes were analyzed. Where poor correlation between stable isotope labeling and spectral counting was found...

Toward improved peptide feature detection in quantitative proteomics using stable isotope labeling.

Science.gov (United States)

Nilse, Lars; Sigloch, Florian Christoph; Biniossek, Martin L; Schilling, Oliver

2015-08-01

Reliable detection of peptides in LC-MS data is a key algorithmic step in the analysis of quantitative proteomics experiments. While highly abundant peptides can be detected reliably by most modern software tools, there is much less agreement on medium and low-intensity peptides in a sample. The choice of software tools can have a big impact on the quantification of proteins, especially for proteins that appear in lower concentrations. However, in many experiments, it is precisely this region of less abundant but substantially regulated proteins that holds the biggest potential for discoveries. This is particularly true for discovery proteomics in the pharmacological sector with a specific interest in key regulatory proteins. In this viewpoint article, we discuss how the development of novel software algorithms allows us to study this region of the proteome with increased confidence. Reliable results are one of many aspects to be considered when deciding on a bioinformatics software platform. Deployment into existing IT infrastructures, compatibility with other software packages, scalability, automation, flexibility, and support need to be considered and are briefly addressed in this viewpoint article. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics reveals the kinetics of trypsin-catalyzed protein digestion.

Science.gov (United States)

Pan, Yanbo; Cheng, Kai; Mao, Jiawei; Liu, Fangjie; Liu, Jing; Ye, Mingliang; Zou, Hanfa

2014-10-01

Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.

Identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method

KAUST Repository

Liu, Pei

2014-01-28

Cellular redox status plays a key role in mediating various physiological and developmental processes often through modulating activities of redox-sensitive proteins. Various stresses trigger over-production of reactive oxygen/nitrogen species which lead to oxidative modifications of redox-sensitive proteins. Identification and characterization of redox-sensitive proteins are important steps toward understanding molecular mechanisms of stress responses. Here, we report a high-throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential labeling of reduced and oxidized thiols. The biotin-tagged peptides are affinity purified, labeled with iTRAQ reagents, and analyzed using a paralleled HCD-CID fragmentation mode in an LTQ-Orbitrap. With this approach, we identified 195 cysteine-containing peptides from 179 proteins whose thiols underwent oxidative modifications in Arabidopsis cells following the treatment with hydrogen peroxide. A majority of those redox-sensitive proteins, including several transcription factors, were not identified by previous redox proteomics studies. This approach allows identification of the specific redox-regulated cysteine residues, and offers an effective tool for elucidation of redox proteomes. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Towards high throughput and spatiotemporal proteomics : analytical workflows and quantitative label-free mass spectrometry

NARCIS (Netherlands)

Mostovenko, Ekaterina

2013-01-01

A large part of modern biology is dedicated to the functional annotation and interpretation of genetic information and its influence on the subject’s phenotype. Proteomics describes the state of the system from the perspective of expression, structure, localization, interaction and function of the

Quantitative proteomics study of larval settlement in the barnacle Balanus amphitrite

KAUST Repository

Chen, Zhang-Fan; Zhang, Huoming; Wang, Hao; Matsumura, Kiyotaka; Wong, Yue Him; Ravasi, Timothy; Qian, Pei-Yuan

2014-01-01

Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite. © 2014 Chen et al.

Quantitative proteomics study of larval settlement in the barnacle Balanus amphitrite

KAUST Repository

Chen, Zhang-Fan

2014-02-13

Barnacles are major sessile components of the intertidal areas worldwide, and also one of the most dominant fouling organisms in fouling communities. Larval settlement has a crucial ecological effect not only on the distribution of the barnacle population but also intertidal community structures. However, the molecular mechanisms involved in the transition process from the larval to the juvenile stage remain largely unclear. In this study, we carried out comparative proteomic profiles of stage II nauplii, stage VI nauplii, cyprids, and juveniles of the barnacle Balanus amphitrite using label-free quantitative proteomics, followed by the measurement of the gene expression levels of candidate proteins. More than 700 proteins were identified at each stage; 80 were significantly up-regulated in cyprids and 95 in juveniles vs other stages. Specifically, proteins involved in energy and metabolism, the nervous system and signal transduction were significantly up-regulated in cyprids, whereas proteins involved in cytoskeletal remodeling, transcription and translation, cell proliferation and differentiation, and biomineralization were up-regulated in juveniles, consistent with changes associated with larval metamorphosis and tissue remodeling in juveniles. These findings provided molecular evidence for the morphological, physiological and biological changes that occur during the transition process from the larval to the juvenile stages in B. amphitrite. © 2014 Chen et al.

Label-Free LC-MS/MS Proteomic Analysis of Cerebrospinal Fluid Identifies Protein/Pathway Alterations and Candidate Biomarkers for Amyotrophic Lateral Sclerosis.

Science.gov (United States)

Collins, Mahlon A; An, Jiyan; Hood, Brian L; Conrads, Thomas P; Bowser, Robert P

2015-11-06

Analysis of the cerebrospinal fluid (CSF) proteome has proven valuable to the study of neurodegenerative disorders. To identify new protein/pathway alterations and candidate biomarkers for amyotrophic lateral sclerosis (ALS), we performed comparative proteomic profiling of CSF from sporadic ALS (sALS), healthy control (HC), and other neurological disease (OND) subjects using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). A total of 1712 CSF proteins were detected and relatively quantified by spectral counting. Levels of several proteins with diverse biological functions were significantly altered in sALS samples. Enrichment analysis was used to link these alterations to biological pathways, which were predominantly related to inflammation, neuronal activity, and extracellular matrix regulation. We then used our CSF proteomic profiles to create a support vector machines classifier capable of discriminating training set ALS from non-ALS (HC and OND) samples. Four classifier proteins, WD repeat-containing protein 63, amyloid-like protein 1, SPARC-like protein 1, and cell adhesion molecule 3, were identified by feature selection and externally validated. The resultant classifier distinguished ALS from non-ALS samples with 83% sensitivity and 100% specificity in an independent test set. Collectively, our results illustrate the utility of CSF proteomic profiling for identifying ALS protein/pathway alterations and candidate disease biomarkers.

Label-free cell-cycle analysis by high-throughput quantitative phase time-stretch imaging flow cytometry

Science.gov (United States)

Mok, Aaron T. Y.; Lee, Kelvin C. M.; Wong, Kenneth K. Y.; Tsia, Kevin K.

2018-02-01

Biophysical properties of cells could complement and correlate biochemical markers to characterize a multitude of cellular states. Changes in cell size, dry mass and subcellular morphology, for instance, are relevant to cell-cycle progression which is prevalently evaluated by DNA-targeted fluorescence measurements. Quantitative-phase microscopy (QPM) is among the effective biophysical phenotyping tools that can quantify cell sizes and sub-cellular dry mass density distribution of single cells at high spatial resolution. However, limited camera frame rate and thus imaging throughput makes QPM incompatible with high-throughput flow cytometry - a gold standard in multiparametric cell-based assay. Here we present a high-throughput approach for label-free analysis of cell cycle based on quantitative-phase time-stretch imaging flow cytometry at a throughput of > 10,000 cells/s. Our time-stretch QPM system enables sub-cellular resolution even at high speed, allowing us to extract a multitude (at least 24) of single-cell biophysical phenotypes (from both amplitude and phase images). Those phenotypes can be combined to track cell-cycle progression based on a t-distributed stochastic neighbor embedding (t-SNE) algorithm. Using multivariate analysis of variance (MANOVA) discriminant analysis, cell-cycle phases can also be predicted label-free with high accuracy at >90% in G1 and G2 phase, and >80% in S phase. We anticipate that high throughput label-free cell cycle characterization could open new approaches for large-scale single-cell analysis, bringing new mechanistic insights into complex biological processes including diseases pathogenesis.

Performance of isobaric and isotopic labeling in quantitative plant proteomics

DEFF Research Database (Denmark)

Nogueira, Fábio C S; Palmisano, Giuseppe; Schwämmle, Veit

2012-01-01

, and quantitation. In the present work, we have used LC-MS to compare an isotopic (ICPL) and isobaric (iTRAQ) chemical labeling technique to quantify proteins in the endosperm of Ricinus communis seeds at three developmental stages (IV, VI, and X). Endosperm proteins of each stage were trypsin-digested in...

Label-Free Quantitative Proteomic Analysis of Harmless and Pathogenic Strains of Infectious Microalgae, Prototheca spp.

Directory of Open Access Journals (Sweden)

Jayaseelan Murugaiyan

2016-12-01

Full Text Available Microalgae of the genus Prototheca (P. spp are associated with rare algal infections of invertebrates termed protothecosis. Among the seven generally accepted species, P. zopfii genotype 2 (GT2 is associated with a severe form of bovine mastitis while P. blaschkeae causes the mild and sub-clinical form of mastitis. The reason behind the infectious nature of P. zopfii GT2, while genotype 1 (GT1 remains non-infectious, is not known. Therefore, in the present study we investigated the protein expression level difference between the genotypes of P. zopfii and P. blaschkeae. Cells were cultured to the mid-exponential phase, harvested, and processed for LC-MS analysis. Peptide data was acquired on an LTQ Orbitrap Velos, raw spectra were quantitatively analyzed with MaxQuant software and matching with the reference database of Chlorella variabilis and Auxenochlorella protothecoides resulted in the identification of 226 proteins. Comparison of an environmental strain with infectious strains resulted in the identification of 51 differentially expressed proteins related to carbohydrate metabolism, energy production and protein translation. The expression level of Hsp70 proteins and their role in the infectious process is worth further investigation. All mass spectrometry data are available via ProteomeXchange with identifier PXD005305.

Inspection, visualisation and analysis of quantitative proteomics data

OpenAIRE

Gatto, Laurent

2016-01-01

Material Quantitative Proteomics and Data Analysis Course. 4 - 5 April 2016, Queen Hotel, Chester, UK Table D - Inspection, visualisation and analysis of quantitative proteomics data, Laurent Gatto (University of Cambridge)

YPED: an integrated bioinformatics suite and database for mass spectrometry-based proteomics research.

Science.gov (United States)

Colangelo, Christopher M; Shifman, Mark; Cheung, Kei-Hoi; Stone, Kathryn L; Carriero, Nicholas J; Gulcicek, Erol E; Lam, TuKiet T; Wu, Terence; Bjornson, Robert D; Bruce, Can; Nairn, Angus C; Rinehart, Jesse; Miller, Perry L; Williams, Kenneth R

2015-02-01

We report a significantly-enhanced bioinformatics suite and database for proteomics research called Yale Protein Expression Database (YPED) that is used by investigators at more than 300 institutions worldwide. YPED meets the data management, archival, and analysis needs of a high-throughput mass spectrometry-based proteomics research ranging from a single laboratory, group of laboratories within and beyond an institution, to the entire proteomics community. The current version is a significant improvement over the first version in that it contains new modules for liquid chromatography-tandem mass spectrometry (LC-MS/MS) database search results, label and label-free quantitative proteomic analysis, and several scoring outputs for phosphopeptide site localization. In addition, we have added both peptide and protein comparative analysis tools to enable pairwise analysis of distinct peptides/proteins in each sample and of overlapping peptides/proteins between all samples in multiple datasets. We have also implemented a targeted proteomics module for automated multiple reaction monitoring (MRM)/selective reaction monitoring (SRM) assay development. We have linked YPED's database search results and both label-based and label-free fold-change analysis to the Skyline Panorama repository for online spectra visualization. In addition, we have built enhanced functionality to curate peptide identifications into an MS/MS peptide spectral library for all of our protein database search identification results. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

Science.gov (United States)

Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

2017-08-01

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

Strigolactone-Regulated Proteins Revealed by iTRAQ-Based Quantitative Proteomics in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Li, Zhou [ORNL; Czarnecki, Olaf [ORNL; Chourey, Karuna [ORNL; Yang, Jun [ORNL; Tuskan, Gerald A [ORNL; Hurst, Gregory {Greg} B [ORNL; Pan, Chongle [ORNL; Chen, Jay [ORNL

2014-01-01

Strigolactones (SLs) are a new class of plant hormones. In addition to acting as a key inhibitor of shoot branching, SLs stimulate seed germination of root parasitic plants and promote hyphal branching and root colonization of symbiotic arbuscular mycorrhizal fungi. They also regulate many other aspects of plant growth and development. At the transcription level, SL-regulated genes have been reported. However, nothing is known about the proteome regulated by this new class of plant hormones. Here, a quantitative proteomics approach using an isobaric chemical labeling reagent, iTRAQ, to identify the proteome regulated by SLs in Arabidopsis seedlings is presented. It was found SLs regulate the expression of about three dozens of proteins that have not been previously assigned to SL pathways. These findings provide a new tool to investigate the molecular mechanism of action of SLs.

Comparative Membrane Proteomics Reveals a Nonannotated E. coli Heat Shock Protein.

Science.gov (United States)

Yuan, Peijia; D'Lima, Nadia G; Slavoff, Sarah A

2018-01-09

Recent advances in proteomics and genomics have enabled discovery of thousands of previously nonannotated small open reading frames (smORFs) in genomes across evolutionary space. Furthermore, quantitative mass spectrometry has recently been applied to analysis of regulated smORF expression. However, bottom-up proteomics has remained relatively insensitive to membrane proteins, suggesting they may have been underdetected in previous studies. In this report, we add biochemical membrane protein enrichment to our previously developed label-free quantitative proteomics protocol, revealing a never-before-identified heat shock protein in Escherichia coli K12. This putative smORF-encoded heat shock protein, GndA, is likely to be ∼36-55 amino acids in length and contains a predicted transmembrane helix. We validate heat shock-regulated expression of the gndA smORF and demonstrate that a GndA-GFP fusion protein cofractionates with the cell membrane. Quantitative membrane proteomics therefore has the ability to reveal nonannotated small proteins that may play roles in bacterial stress responses.

Recent advances on multidimensional liquid chromatography–mass spectrometry for proteomics: From qualitative to quantitative analysis—A review

International Nuclear Information System (INIS)

Wu Qi; Yuan Huiming; Zhang Lihua; Zhang Yukui

2012-01-01

Highlights: ► We discuss progress of MDLC–MS systems in qualitative and quantitative proteomics. ► Both “Top-down” and “bottom-up” strategies are discussed in detail. ► On-line integrations of stable isotope labeling process are highlighted. ► This review gives insights into further directions for higher level integration. - Abstract: With the acceleration of proteome research, increasing attention has been paid to multidimensional liquid chromatography–mass spectrometry (MDLC–MS) due to its high peak capacity and separation efficiency. Recently, many efforts have been put to improve MDLC-based strategies including “top-down” and “bottom-up” to enable highly sensitive qualitative and quantitative analysis of proteins, as well as accelerate the whole analytical procedure. Integrated platforms with combination of sample pretreatment, multidimensional separations and identification were also developed to achieve high throughput and sensitive detection of proteomes, facilitating highly accurate and reproducible quantification. This review summarized the recent advances of such techniques and their applications in qualitative and quantitative analysis of proteomes.

Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

KAUST Repository

Turek, Ilona; Wheeler, Janet I.; Gehring, Christoph A; Irving, Helen R.; Marondedze, Claudius

2015-01-01

Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

Quantitative proteome changes in Arabidopsis thaliana suspension-cultured cells in response to plant natriuretic peptides

KAUST Repository

Turek, Ilona

2015-06-30

Proteome changes in the Arabidopsis thaliana suspension cells in response to the A. thaliana plant natriuretic peptide (PNP), AtPNP-A (At2g18660) were assessed using quantitative proteomics employing tandem mass tag (TMT) labeling and tandem mass spectrometry (LC–MS/MS). In this study, we characterized temporal responses of suspension-cultured cells to 1 nM and 10 pM AtPNP-A at 0, 10 and 30 min post-treatment. Both concentrations we found to yield a distinct differential proteome signature. The data shown in this article are associated with the article “Plant natriuretic peptides induce a specific set of proteins diagnostic for an adaptive response to abiotic stress” by Turek et al. (Front. Plant Sci. 5 (2014) 661) and have been deposited to the ProteomeXchange with identifier PXD001386.

Quantitative proteome analysis of plasma microparticles for the characterization of HCV-induced hepatic cirrhosis and hepatocellular carcinoma.

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Taleb, Raghda Saad Zaghloul; Moez, Pacint; Younan, Doreen; Eisenacher, Martin; Tenbusch, Matthias; Sitek, Barbara; Bracht, Thilo

2017-12-01

Hepatocellular carcinoma (HCC) is the most common primary malignant liver tumor and a leading cause of cancer-related deaths worldwide. Cirrhosis induced by hepatitis-C virus (HCV) infection is the most critical risk factor for HCC. However, the mechanism of HCV-induced carcinogenesis is not fully understood. Plasma microparticles (PMP) contribute to numerous physiological and pathological processes and contain proteins whose composition correlates to the respective pathophysiological conditions. We analyzed PMP from 22 HCV-induced cirrhosis patients, 16 HCV-positive HCC patients with underlying cirrhosis and 18 healthy controls. PMP were isolated using ultracentrifugation and analyzed via label-free LC-MS/MS. We identified 840 protein groups and quantified 507 proteins. 159 proteins were found differentially abundant between the three experimental groups. PMP in both disease entities displayed remarkable differences in the proteome composition compared to healthy controls. Conversely, the proteome difference between both diseases was minimal. GO analysis revealed that PMP isolated from both diseases were significantly enriched in proteins involved in complement activation, while endopeptidase activity was downregulated exclusively in HCC patients. This study reports for the first time a quantitative proteome analysis for PMP from patients with HCV-induced cirrhosis and HCC. Data are available via ProteomeXchange with identifier PXD005777. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Detection of Nuclear Protein Profile Changes by Human Metapneumovirus M2-2 Protein Using Quantitative Differential Proteomics

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Yuping Ren

2017-12-01

Full Text Available Human metapneumovirus (hMPV is a leading cause of lower respiratory infection in pediatric populations globally. This study examined proteomic profile changes in A549 cells infected with hMPV and two attenuated mutants with deleted PDZ domain-binding motif(s in the M2-2 protein. These motifs are involved in the interruption of antiviral signaling, namely the interaction between the TNF receptor associated factor (TRAF and mitochondrial antiviral-signaling (MAVS proteins. The aim of this study was to provide insight into the overall and novel impact of M2-2 motifs on cellular responses via an unbiased comparison. Tandem mass tagging, stable isotope labeling, and high-resolution mass spectrometry were used for quantitative proteomic analysis. Using quantitative proteomics and Venn analysis, 1248 common proteins were detected in all infected samples of both technical sets. Hierarchical clustering of the differentiated proteome displayed distinct proteomic signatures that were controlled by the motif(s. Bioinformatics and experimental analysis confirmed the differentiated proteomes, revealed novel cellular biological events, and implicated key pathways controlled by hMPV M2-2 PDZ domain-binding motif(s. This provides further insight for evaluating M2-2 mutants as potent vaccine candidates.

Quantitative Proteomics Analysis of Altered Protein Expression in the Placental Villous Tissue of Early Pregnancy Loss Using Isobaric Tandem Mass Tags

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Xiaobei Ni

2014-01-01

Full Text Available Many pregnant women suffer miscarriages during early gestation, but the description of these early pregnancy losses (EPL can be somewhat confusing because of the complexities of early development. Thus, the identification of proteins with different expression profiles related to early pregnancy loss is essential for understanding the comprehensive pathophysiological mechanism. In this study, we report a gel-free tandem mass tags- (TMT- labeling based proteomic analysis of five placental villous tissues from patients with early pregnancy loss and five from normal pregnant women. The application of this method resulted in the identification of 3423 proteins and 19647 peptides among the patient group and the matched normal control group. Qualitative and quantitative proteomic analysis revealed 51 proteins to be differentially abundant between the two groups (≥1.2-fold, Student's t-test, P<0.05. To obtain an overview of the biological functions of the proteins whose expression levels altered significantly in EPL group, gene ontology analysis was performed. We also investigated the twelve proteins with a difference over 1.5-fold using pathways analysis. Our results demonstrate that the gel-free TMT-based proteomic approach allows the quantification of differences in protein expression levels, which is useful for obtaining molecular insights into early pregnancy loss.

Quantitative Peptidomics with Five-plex Reductive Methylation labels

Science.gov (United States)

Tashima, Alexandre K.; Fricker, Lloyd D.

2018-05-01

Quantitative peptidomics and proteomics often use chemical tags to covalently modify peptides with reagents that differ in the number of stable isotopes, allowing for quantitation of the relative peptide levels in the original sample based on the peak height of each isotopic form. Different chemical reagents have been used as tags for quantitative peptidomics and proteomics, and all have strengths and weaknesses. One of the simplest approaches uses formaldehyde and sodium cyanoborohydride to methylate amines, converting primary and secondary amines into tertiary amines. Up to five different isotopic forms can be generated, depending on the isotopic forms of formaldehyde and cyanoborohydride reagents, allowing for five-plex quantitation. However, the mass difference between each of these forms is only 1 Da per methyl group incorporated into the peptide, and for many peptides there is substantial overlap from the natural abundance of 13C and other isotopes. In this study, we calculated the contribution from the natural isotopes for 26 native peptides and derived equations to correct the peak intensities. These equations were applied to data from a study using human embryonic kidney HEK293T cells in which five replicates were treated with 100 nM vinblastine for 3 h and compared with five replicates of cells treated with control medium. The correction equations brought the replicates to the expected 1:1 ratios and revealed significant decreases in levels of 21 peptides upon vinblastine treatment. These equations enable accurate quantitation of small changes in peptide levels using the reductive methylation labeling approach. [Figure not available: see fulltext.

Quantitative proteomic view on secreted, cell surface-associated, and cytoplasmic proteins of the methicillin-resistant human pathogen Staphylococcus aureus under iron-limited conditions.

Science.gov (United States)

Hempel, Kristina; Herbst, Florian-Alexander; Moche, Martin; Hecker, Michael; Becher, Dörte

2011-04-01

Staphylococcus aureus is capable of colonizing and infecting humans by its arsenal of surface-exposed and secreted proteins. Iron-limited conditions in mammalian body fluids serve as a major environmental signal to bacteria to express virulence determinants. Here we present a comprehensive, gel-free, and GeLC-MS/MS-based quantitative proteome profiling of S. aureus under this infection-relevant situation. (14)N(15)N metabolic labeling and three complementing approaches were combined for relative quantitative analyses of surface-associated proteins. The surface-exposed and secreted proteome profiling approaches comprise trypsin shaving, biotinylation, and precipitation of the supernatant. By analysis of the outer subproteomic and cytoplasmic protein fraction, 1210 proteins could be identified including 221 surface-associated proteins. Thus, access was enabled to 70% of the predicted cell wall-associated proteins, 80% of the predicted sortase substrates, two/thirds of lipoproteins and more than 50% of secreted and cytoplasmic proteins. For iron-deficiency, 158 surface-associated proteins were quantified. Twenty-nine proteins were found in altered amounts showing particularly surface-exposed proteins strongly induced, such as the iron-regulated surface determinant proteins IsdA, IsdB, IsdC and IsdD as well as lipid-anchored iron compound-binding proteins. The work presents a crucial subject for understanding S. aureus pathophysiology by the use of methods that allow quantitative surface proteome profiling.

Quantitative Label-Free Cell Proliferation Tracking with a Versatile Electrochemical Impedance Detection Platform

DEFF Research Database (Denmark)

Caviglia, Claudia; Carminati, M; Heiskanen, Arto

2012-01-01

optimal detection strategies. Electrochemical Impedance Spectroscopy (EIS) has been used to monitor and compare adhesion of different cell lines. HeLa cells and 3T3 fibroblasts have been cultured for 12 hours on interdigitated electrode arrays integrated into a tailor-made cell culture platform. Both......Since the use of impedance measurements for label-free monitoring of cells has become widespread but still the choice of sensing configuration is not unique though crucial for a quantitative interpretation of data, we demonstrate the application of a novel custom multipotentiostat platform to study...... vertical and coplanar interdigitated sensing configuration approaches have been used and compared on the same cell populations....

Method and platform standardization in MRM-based quantitative plasma proteomics.

Science.gov (United States)

Percy, Andrew J; Chambers, Andrew G; Yang, Juncong; Jackson, Angela M; Domanski, Dominik; Burkhart, Julia; Sickmann, Albert; Borchers, Christoph H

2013-12-16

There exists a growing demand in the proteomics community to standardize experimental methods and liquid chromatography-mass spectrometry (LC/MS) platforms in order to enable the acquisition of more precise and accurate quantitative data. This necessity is heightened by the evolving trend of verifying and validating candidate disease biomarkers in complex biofluids, such as blood plasma, through targeted multiple reaction monitoring (MRM)-based approaches with stable isotope-labeled standards (SIS). Considering the lack of performance standards for quantitative plasma proteomics, we previously developed two reference kits to evaluate the MRM with SIS peptide approach using undepleted and non-enriched human plasma. The first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). Here, these kits have been refined for practical use and then evaluated through intra- and inter-laboratory testing on 6 common LC/MS platforms. For an identical panel of 22 plasma proteins, similar concentrations were determined, regardless of the kit, instrument platform, and laboratory of analysis. These results demonstrate the value of the kit and reinforce the utility of standardized methods and protocols. The proteomics community needs standardized experimental protocols and quality control methods in order to improve the reproducibility of MS-based quantitative data. This need is heightened by the evolving trend for MRM-based validation of proposed disease biomarkers in complex biofluids such as blood plasma. We have developed two kits to assist in the inter- and intra-laboratory quality control of MRM experiments: the first kit tests the effectiveness of the LC/MRM-MS platform (kit #1), while the second evaluates the performance of an entire analytical workflow (kit #2). In this paper, we report the use of these kits in intra- and inter-laboratory testing on 6 common LC/MS platforms. This

Serum proteome profiling in canine idiopathic dilated cardiomyopathy using TMT-based quantitative proteomics approach.

Science.gov (United States)

Bilić, Petra; Guillemin, Nicolas; Kovačević, Alan; Beer Ljubić, Blanka; Jović, Ines; Galan, Asier; Eckersall, Peter David; Burchmore, Richard; Mrljak, Vladimir

2018-05-15

Idiopathic dilated cardiomyopathy (iDCM) is a primary myocardial disorder with an unknown aetiology, characterized by reduced contractility and ventricular dilation of the left or both ventricles. Naturally occurring canine iDCM was used herein to identify serum proteomic signature of the disease compared to the healthy state, providing an insight into underlying mechanisms and revealing proteins with biomarker potential. To achieve this, we used high-throughput label-based quantitative LC-MS/MS proteomics approach and bioinformatics analysis of the in silico inferred interactome protein network created from the initial list of differential proteins. To complement the proteomic analysis, serum biochemical parameters and levels of know biomarkers of cardiac function were measured. Several proteins with biomarker potential were identified, such as inter-alpha-trypsin inhibitor heavy chain H4, microfibril-associated glycoprotein 4 and apolipoprotein A-IV, which were validated using an independent method (Western blotting) and showed high specificity and sensitivity according to the receiver operating characteristic curve analysis. Bioinformatics analysis revealed involvement of different pathways in iDCM, such as complement cascade activation, lipoprotein particles dynamics, elastic fibre formation, GPCR signalling and respiratory electron transport chain. Idiopathic dilated cardiomyopathy is a severe primary myocardial disease of unknown cause, affecting both humans and dogs. This study is a contribution to the canine heart disease research by means of proteomic and bioinformatic state of the art analyses, following similar approach in human iDCM research. Importantly, we used serum as non-invasive and easily accessible biological source of information and contributed to the scarce data on biofluid proteome research on this topic. Bioinformatics analysis revealed biological pathways modulated in canine iDCM with potential of further targeted research. Also, several

Proteomic Identification and Quantification of S-glutathionylation in Mouse Macrophages Using Resin-Assisted Enrichment and Isobaric Labeling

Energy Technology Data Exchange (ETDEWEB)

Su, Dian; Gaffrey, Matthew J.; Guo, Jia; Hatchell, Kayla E.; Chu, Rosalie K.; Clauss, Therese RW; Aldrich, Joshua T.; Wu, Si; Purvine, Samuel O.; Camp, David G.; Smith, Richard D.; Thrall, Brian D.; Qian, Weijun

2014-02-11

Protein S-glutathionylation (SSG) is an important regulatory posttranslational modification of protein cysteine (Cys) thiol redox switches, yet the role of specific cysteine residues as targets of modification is poorly understood. We report a novel quantitative mass spectrometry (MS)-based proteomic method for site-specific identification and quantification of S-glutathionylation across different conditions. Briefly, this approach consists of initial blocking of free thiols by alkylation, selective reduction of glutathionylated thiols and enrichment using thiol affinity resins, followed by on-resin tryptic digestion and isobaric labeling with iTRAQ (isobaric tags for relative and absolute quantitation) for MS-based identification and quantification. The overall approach was validated by application to RAW 264.7 mouse macrophages treated with different doses of diamide to induce glutathionylation. A total of 1071 Cys-sites from 690 proteins were identified in response to diamide treatment, with ~90% of the sites displaying >2-fold increases in SSG-modification compared to controls.. This approach was extended to identify potential SSG modified Cys-sites in response to H2O2, an endogenous oxidant produced by activated macrophages and many pathophysiological stimuli. The results revealed 364 Cys-sites from 265 proteins that were sensitive to S-glutathionylation in response to H2O2 treatment. These proteins covered a range of molecular types and molecular functions with free radical scavenging, and cell death and survival included as the most significantly enriched functional categories. Overall the results demonstrate that our approach is effective for site-specific identification and quantification of S-glutathionylated proteins. The analytical strategy also provides a unique approach to determining the major pathways and cell processes most susceptible to glutathionylation at a proteome-wide scale.

Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses.

Science.gov (United States)

Zai, Xiaodong; Yang, Qiaoling; Yin, Ying; Li, Ruihua; Qian, Mengying; Zhao, Taoran; Li, Yaohui; Zhang, Jun; Fu, Ling; Xu, Junjie; Chen, Wei

2017-01-01

Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS) via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs) that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular homeostasis and metabolic

Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana

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Giuseppe Dionisio

2016-10-01

Full Text Available Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w/v of dead intact American cockroach (Periplaneta americana or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion.

Label-Free Differential Proteomics and Quantification of Exoenzymes from Isolates of the Entomopathogenic Fungus Beauveria bassiana.

Science.gov (United States)

Dionisio, Giuseppe; Kryger, Per; Steenberg, Tove

2016-10-14

Beauveria bassiana is an entomopathogenic fungus that grows both in vivo and in vitro. In vivo it can colonize live insect hosts, and tissue digestion occurs by secreted hydrolytic exoenzymes. It can also colonize dead insect tissue provided this is free from competing microorganisms. Depending on whether the host is alive or dead the expression (quality/quantity) of the exoenzymes may vary. We have grown several isolates of B. bassiana in shaking flasks for 120 h at 25 °C in order to evaluate the maximal exoenzyme production using two diet regimes. As sole carbon, nitrogen, and phosphate sources we used 1% shrimp chitin and either 0.5% w / v of dead intact American cockroach ( Periplaneta americana ) or their isolated cuticles. This is the first report of a differential proteomics of B. bassiana exoenzymes performed by label-free nano-LC MS/MS. Total proteolytic enzyme activity was mainly due to Pr1A or Pr1B depending on the isolate and the diet regime. The most differentially secreted enzymes were: the cuticle-degrading subtilisin Pr1A, GH13 alpha-glycosidase, glucan endo-1,3-beta-glucosidase, subtilisin-like proteinase Spm1, lipase 1, beta-1,3 exoglucanase, and endo-1,3-beta-glucosidase. Among the B. bassiana isolates analyzed, Bb 678 and Bb BG were the most active in Pr1A secretion.

PIQMIe: a web server for semi-quantitative proteomics data management and analysis.

Science.gov (United States)

Kuzniar, Arnold; Kanaar, Roland

2014-07-01

We present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates peptide and (non-redundant) protein identifications and quantitations from multiple experiments with additional biological information on the protein entries, and makes the linked data available in the form of a light-weight relational database, which enables dedicated data analyses (e.g. in R) and user-driven queries. Using the web interface, users are presented with a concise summary of their proteomics experiments in numerical and graphical forms, as well as with a searchable protein grid and interactive visualization tools to aid in the rapid assessment of the experiments and in the identification of proteins of interest. The web server not only provides data access through a web interface but also supports programmatic access through RESTful web service. The web server is available at http://piqmie.semiqprot-emc.cloudlet.sara.nl or http://www.bioinformatics.nl/piqmie. This website is free and open to all users and there is no login requirement. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

A comparison of labeling and label-free mass spectrometry-based proteomics approaches.

Science.gov (United States)

Patel, Vibhuti J; Thalassinos, Konstantinos; Slade, Susan E; Connolly, Joanne B; Crombie, Andrew; Murrell, J Colin; Scrivens, James H

2009-07-01

The proteome of the recently discovered bacterium Methylocella silvestris has been characterized using three profiling and comparative proteomics approaches. The organism has been grown on two different substrates enabling variations in protein expression to be identified. The results obtained using the experimental approaches have been compared with respect to number of proteins identified, confidence in identification, sequence coverage and agreement of regulated proteins. The sample preparation, instrumental time and sample loading requirements of the differing experiments are compared and discussed. A preliminary screen of the protein regulation results for biological significance has also been performed.

RAPID AND AUTOMATED PROCESSING OF MALDI-FTICR/MS DATA FOR N-METABOLIC LABELING IN A SHOTGUN PROTEOMICS ANALYSIS.

Science.gov (United States)

Jing, Li; Amster, I Jonathan

2009-10-15

Offline high performance liquid chromatography combined with matrix assisted laser desorption and Fourier transform ion cyclotron resonance mass spectrometry (HPLC-MALDI-FTICR/MS) provides the means to rapidly analyze complex mixtures of peptides, such as those produced by proteolytic digestion of a proteome. This method is particularly useful for making quantitative measurements of changes in protein expression by using (15)N-metabolic labeling. Proteolytic digestion of combined labeled and unlabeled proteomes produces complex mixtures that with many mass overlaps when analyzed by HPLC-MALDI-FTICR/MS. A significant challenge to data analysis is the matching of pairs of peaks which represent an unlabeled peptide and its labeled counterpart. We have developed an algorithm and incorporated it into a compute program which significantly accelerates the interpretation of (15)N metabolic labeling data by automating the process of identifying unlabeled/labeled peak pairs. The algorithm takes advantage of the high resolution and mass accuracy of FTICR mass spectrometry. The algorithm is shown to be able to successfully identify the (15)N/(14)N peptide pairs and calculate peptide relative abundance ratios in highly complex mixtures from the proteolytic digest of a whole organism protein extract.

Redox proteomic analysis of the gastrocnemius muscle from adult and old mice

Directory of Open Access Journals (Sweden)

Brian McDonagh

2015-09-01

Full Text Available The data provides information in support of the research article, “Differential Cysteine Labeling and Global Label-Free Proteomics Reveals an Altered Metabolic State in Skeletal Muscle Aging”, Journal of Proteome Research, 2014, 13 (11, 2008–21 [1]. Raw data is available from ProteomeXchange [2] with identifier PDX001054. The proteome of gastrocnemius muscle from adult and old mice was analyzed by global label-free proteomics and the relative quantification of specific reduced and reversibly oxidized Cysteine (Cys residues was performed using Skyline [3]. Briefly, reduced Cysteine (Cys containing peptides was alkylated using N-ethylmalemide (d0-NEM. Samples were desalted and reversibly oxidized Cys residues were reduced using tris(2-carboxyethylphosphine (TCEP and the newly formed reduced Cys residues were labeled with heavy NEM( d5-NEM. Label-free analysis of the global proteome of adult (n=5 and old (n=4 gastrocnemius muscles was performed using Peaks7™ mass spectrometry data analysis software [4]. Relative quantification of Cys containing peptides that were identified as reduced (d(0 NEM labeled and reversibly oxidized d(5–NEM labeled was performed using the intensity of their precursor ions in Skyline. Results indicate that muscles from old mice show reduced redox flexibility particularly in proteins involved in the generation of precursor metabolites and energy metabolism, indicating a loss in the flexibility of the redox energy response.

Quantitative Proteomic Analysis of the Response to Zinc, Magnesium, and Calcium Deficiency in Specific Cell Types of Arabidopsis Roots

Directory of Open Access Journals (Sweden)

Yoichiro Fukao

2016-01-01

Full Text Available The proteome profiles of specific cell types have recently been investigated using techniques such as fluorescence activated cell sorting and laser capture microdissection. However, quantitative proteomic analysis of specific cell types has not yet been performed. In this study, to investigate the response of the proteome to zinc, magnesium, and calcium deficiency in specific cell types of Arabidopsis thaliana roots, we performed isobaric tags for relative and absolute quantification (iTRAQ-based quantitative proteomics using GFP-expressing protoplasts collected by fluorescence-activated cell sorting. Protoplasts were collected from the pGL2-GFPer and pMGP-GFPer marker lines for epidermis or inner cell lines (pericycle, endodermis, and cortex, respectively. To increase the number of proteins identified, iTRAQ-labeled peptides were separated into 24 fractions by OFFGFEL electrophoresis prior to high-performance liquid chromatography coupled with mass spectrometry analysis. Overall, 1039 and 737 proteins were identified and quantified in the epidermal and inner cell lines, respectively. Interestingly, the expression of many proteins was decreased in the epidermis by mineral deficiency, although a weaker effect was observed in inner cell lines such as the pericycle, endodermis, and cortex. Here, we report for the first time the quantitative proteomics of specific cell types in Arabidopsis roots.

Quantitative proteomic analysis of paired colorectal cancer and non-tumorigenic tissues reveals signature proteins and perturbed pathways involved in CRC progression and metastasis.

Science.gov (United States)

Sethi, Manveen K; Thaysen-Andersen, Morten; Kim, Hoguen; Park, Cheol Keun; Baker, Mark S; Packer, Nicolle H; Paik, Young-Ki; Hancock, William S; Fanayan, Susan

2015-08-03

Modern proteomics has proven instrumental in our understanding of the molecular deregulations associated with the development and progression of cancer. Herein, we profile membrane-enriched proteome of tumor and adjacent normal tissues from eight CRC patients using label-free nanoLC-MS/MS-based quantitative proteomics and advanced pathway analysis. Of the 948 identified proteins, 184 proteins were differentially expressed (P1.5) between the tumor and non-tumor tissue (69 up-regulated and 115 down-regulated in tumor tissues). The CRC tumor and non-tumor tissues clustered tightly in separate groups using hierarchical cluster analysis of the differentially expressed proteins, indicating a strong CRC-association of this proteome subset. Specifically, cancer associated proteins such as FN1, TNC, DEFA1, ITGB2, MLEC, CDH17, EZR and pathways including actin cytoskeleton and RhoGDI signaling were deregulated. Stage-specific proteome signatures were identified including up-regulated ribosomal proteins and down-regulated annexin proteins in early stage CRC. Finally, EGFR(+) CRC tissues showed an EGFR-dependent down-regulation of cell adhesion molecules, relative to EGFR(-) tissues. Taken together, this study provides a detailed map of the altered proteome and associated protein pathways in CRC, which enhances our mechanistic understanding of CRC biology and opens avenues for a knowledge-driven search for candidate CRC protein markers. Copyright © 2015 Elsevier B.V. All rights reserved.

A Map of General and Specialized Chromatin Readers in Mouse Tissues Generated by Label-free Interaction Proteomics

DEFF Research Database (Denmark)

Eberl, H.C.; Mann, M.; Spruijt, C.G.

2013-01-01

Posttranslational modifications on core histones can serve as binding scaffolds for chromatin-associated proteins. Proteins that specifically bind to or "read" these modifications were previously identified in mass spectrometry-based proteomics screens based on stable isotope-labeling in cell lines...... the chromatin interaction landscape in mouse tissues, our workflow can be used for peptides with different modifications and cell types of any organism....

Large-scale label-free comparative proteomics analysis of polo-like kinase 1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in BRAF(V600E) mutant melanoma cells.

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Cholewa, Brian D; Pellitteri-Hahn, Molly C; Scarlett, Cameron O; Ahmad, Nihal

2014-11-07

Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays a key role during the cell cycle by regulating mitotic entry, progression, and exit. Plk1 is overexpressed in a variety of human cancers and is essential to sustained oncogenic proliferation, thus making Plk1 an attractive therapeutic target. However, the clinical efficacy of Plk1 inhibition has not emulated the preclinical success, stressing an urgent need for a better understanding of Plk1 signaling. This study addresses that need by utilizing a quantitative proteomics strategy to compare the proteome of BRAF(V600E) mutant melanoma cells following treatment with the Plk1-specific inhibitor BI 6727. Employing label-free nano-LC-MS/MS technology on a Q-exactive followed by SIEVE processing, we identified more than 20 proteins of interest, many of which have not been previously associated with Plk1 signaling. Here we report the down-regulation of multiple metabolic proteins with an associated decrease in cellular metabolism, as assessed by lactate and NAD levels. Furthermore, we have also identified the down-regulation of multiple proteasomal subunits, resulting in a significant decrease in 20S proteasome activity. Additionally, we have identified a novel association between Plk1 and p53 through heterogeneous ribonucleoprotein C1/C2 (hnRNPC), thus providing valuable insight into Plk1's role in cancer cell survival.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS.

Science.gov (United States)

Zhang, Hong-Hai; Lechuga, Thomas J; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-05-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. © 2016 by the Society for the Study of Reproduction

The Cytotoxicity Mechanism of 6-Shogaol-Treated HeLa Human Cervical Cancer Cells Revealed by Label-Free Shotgun Proteomics and Bioinformatics Analysis

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Qun Liu

2012-01-01

Full Text Available Cervical cancer is one of the most common cancers among women in the world. 6-Shogaol is a natural compound isolated from the rhizome of ginger (Zingiber officinale. In this paper, we demonstrated that 6-shogaol induced apoptosis and G2/M phase arrest in human cervical cancer HeLa cells. Endoplasmic reticulum stress and mitochondrial pathway were involved in 6-shogaol-mediated apoptosis. Proteomic analysis based on label-free strategy by liquid chromatography chip quadrupole time-of-flight mass spectrometry was subsequently proposed to identify, in a non-target-biased manner, the molecular changes in cellular proteins in response to 6-shogaol treatment. A total of 287 proteins were differentially expressed in response to 24 h treatment with 15 μM 6-shogaol in HeLa cells. Significantly changed proteins were subjected to functional pathway analysis by multiple analyzing software. Ingenuity pathway analysis (IPA suggested that 14-3-3 signaling is a predominant canonical pathway involved in networks which may be significantly associated with the process of apoptosis and G2/M cell cycle arrest induced by 6-shogaol. In conclusion, this work developed an unbiased protein analysis strategy by shotgun proteomics and bioinformatics analysis. Data observed provide a comprehensive analysis of the 6-shogaol-treated HeLa cell proteome and reveal protein alterations that are associated with its anticancer mechanism.

In-depth evaluation of software tools for data-independent acquisition based label-free quantification.

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Kuharev, Jörg; Navarro, Pedro; Distler, Ute; Jahn, Olaf; Tenzer, Stefan

2015-09-01

Label-free quantification (LFQ) based on data-independent acquisition workflows currently experiences increasing popularity. Several software tools have been recently published or are commercially available. The present study focuses on the evaluation of three different software packages (Progenesis, synapter, and ISOQuant) supporting ion mobility enhanced data-independent acquisition data. In order to benchmark the LFQ performance of the different tools, we generated two hybrid proteome samples of defined quantitative composition containing tryptically digested proteomes of three different species (mouse, yeast, Escherichia coli). This model dataset simulates complex biological samples containing large numbers of both unregulated (background) proteins as well as up- and downregulated proteins with exactly known ratios between samples. We determined the number and dynamic range of quantifiable proteins and analyzed the influence of applied algorithms (retention time alignment, clustering, normalization, etc.) on quantification results. Analysis of technical reproducibility revealed median coefficients of variation of reported protein abundances below 5% for MS(E) data for Progenesis and ISOQuant. Regarding accuracy of LFQ, evaluation with synapter and ISOQuant yielded superior results compared to Progenesis. In addition, we discuss reporting formats and user friendliness of the software packages. The data generated in this study have been deposited to the ProteomeXchange Consortium with identifier PXD001240 (http://proteomecentral.proteomexchange.org/dataset/PXD001240). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Salt stress induces changes in the proteomic profile of micropropagated sugarcane shoots

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Reis, Ricardo S.; Heringer, Angelo S.; Rangel, Patricia L.; Santa-Catarina, Claudete; Grativol, Clícia; Veiga, Carlos F. M.; Souza-Filho, Gonçalo A.

2017-01-01

Salt stress is one of the most common stresses in agricultural regions worldwide. In particular, sugarcane is affected by salt stress conditions, and no sugarcane cultivar presently show high productivity accompanied by a tolerance to salt stress. Proteomic analysis allows elucidation of the important pathways involved in responses to various abiotic stresses at the biochemical and molecular levels. Thus, this study aimed to analyse the proteomic effects of salt stress in micropropagated shoots of two sugarcane cultivars (CB38-22 and RB855536) using a label-free proteomic approach. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD006075. The RB855536 cultivar is more tolerant to salt stress than CB38-22. A quantitative label-free shotgun proteomic analysis identified 1172 non-redundant proteins, and 1160 of these were observed in both cultivars in the presence or absence of NaCl. Compared with CB38-22, the RB855536 cultivar showed a greater abundance of proteins involved in non-enzymatic antioxidant mechanisms, ion transport, and photosynthesis. Some proteins, such as calcium-dependent protein kinase, photosystem I, phospholipase D, and glyceraldehyde-3-phosphate dehydrogenase, were more abundant in the RB855536 cultivar under salt stress. Our results provide new insights into the response of sugarcane to salt stress, and the changes in the abundance of these proteins might be important for the acquisition of ionic and osmotic homeostasis during exposure to salt stress. PMID:28419154

Characterization of ubiquitination dependent dynamics in growth factor receptor signaling by quantitative proteomics

DEFF Research Database (Denmark)

Akimov, Vyacheslav; Rigbolt, Kristoffer T G; Nielsen, Mogens M

2011-01-01

Protein ubiquitination is a dynamic reversible post-translational modification that plays a key role in the regulation of numerous cellular processes including signal transduction, endocytosis, cell cycle control, DNA repair and gene transcription. The conjugation of the small protein ubiquitin...... investigating ubiquitination on a proteomic scale, mainly due to the inherited complexity and heterogeneity of ubiquitination. We describe here a quantitative proteomics strategy based on the specificity of ubiquitin binding domains (UBDs) and Stable Isotope Labeling by Amino acids in Cell culture (SILAC...... as ubiquitination-dependent events in signaling pathways. In addition to a detailed seven time-point profile of EGFR ubiquitination over 30 minutes of ligand stimulation, our data determined prominent involvement of Lysine-63 ubiquitin branching in EGF signaling. Furthermore, we found two centrosomal proteins, PCM1...

Quantitative proteomics of Spodoptera frugiperda cells during growth and baculovirus infection.

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Nuno Carinhas

Full Text Available Baculovirus infection of Spodoptera frugiperda cells is a system of choice to produce a range of recombinant proteins, vaccines and, potentially, gene therapy vectors. While baculovirus genomes are well characterized, the genome of S. frugiperda is not sequenced and the virus-host molecular interplay is sparsely known. Herein, we describe the application of stable isotope labeling by amino acids in cell culture (SILAC to obtain the first comparative proteome quantitation of S. frugiperda cells during growth and early baculovirus infection. The proteome coverage was maximized by compiling a search database with protein annotations from insect species. Of interest were differentially proteins related to energy metabolism, endoplasmic reticulum and oxidative stress, yet not investigated in the scope of baculovirus infection. Further, the reduced expression of key viral-encoded proteins early in the infection cycle is suggested to be related with decreased viral replication at high cell density culture. These findings have implications for virological research and improvement of baculovirus-based bioprocesses.

Quantitative Proteomics Analysis of VEGF-Responsive Endothelial Protein S-Nitrosylation Using Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) and LC-MS/MS1

Science.gov (United States)

Zhang, Hong-Hai; Lechuga, Thomas J.; Chen, Yuezhou; Yang, Yingying; Huang, Lan; Chen, Dong-Bao

2016-01-01

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with “light” (L-12C614N4-Arg and L-12C614N2-Lys) or “heavy” (L-13C615N4-Arg and L-13C615N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis. PMID:27075618

In vivo, label-free, three-dimensional quantitative imaging of liver surface using multi-photon microscopy

Energy Technology Data Exchange (ETDEWEB)

Zhuo, Shuangmu, E-mail: shuangmuzhuo@gmail.com, E-mail: hanry-yu@nuhs.edu.sg [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Laser and Optoelectronics Technology, Fujian Normal University, Fuzhou 350007 (China); Yan, Jie [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 14 Medical Drive, MD 11 #04-01A, 117599 Singapore (Singapore); Kang, Yuzhan [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Xu, Shuoyu [Biosystems and Micromechanics IRG, Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14 Enterprise Wing, 138602 Singapore (Singapore); Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Peng, Qiwen [Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, #04-01, 138669 Singapore (Singapore); Computation and System Biology Program, Singapore-MIT Alliance, 4 Engineering Drive 3, E4-04-10, 117576 Singapore (Singapore); Mechanobiology Institute, 5A Engineering Drive 1, T-Lab #05-01, 117411 Singapore (Singapore); and others

2014-07-14

Various structural features on the liver surface reflect functional changes in the liver. The visualization of these surface features with molecular specificity is of particular relevance to understanding the physiology and diseases of the liver. Using multi-photon microscopy (MPM), we have developed a label-free, three-dimensional quantitative and sensitive method to visualize various structural features of liver surface in living rat. MPM could quantitatively image the microstructural features of liver surface with respect to the sinuosity of collagen fiber, the elastic fiber structure, the ratio between elastin and collagen, collagen content, and the metabolic state of the hepatocytes that are correlative with the pathophysiologically induced changes in the regions of interest. This study highlights the potential of this technique as a useful tool for pathophysiological studies and possible diagnosis of the liver diseases with further development.

On the Reproducibility of Label-Free Quantitative Cross-Linking/Mass Spectrometry

Science.gov (United States)

Müller, Fränze; Fischer, Lutz; Chen, Zhuo Angel; Auchynnikava, Tania; Rappsilber, Juri

2018-02-01

Quantitative cross-linking/mass spectrometry (QCLMS) is an emerging approach to study conformational changes of proteins and multi-subunit complexes. Distinguishing protein conformations requires reproducibly identifying and quantifying cross-linked peptides. Here we analyzed the variation between multiple cross-linking reactions using bis[sulfosuccinimidyl] suberate (BS3)-cross-linked human serum albumin (HSA) and evaluated how reproducible cross-linked peptides can be identified and quantified by LC-MS analysis. To make QCLMS accessible to a broader research community, we developed a workflow that integrates the established software tools MaxQuant for spectra preprocessing, Xi for cross-linked peptide identification, and finally Skyline for quantification (MS1 filtering). Out of the 221 unique residue pairs identified in our sample, 124 were subsequently quantified across 10 analyses with coefficient of variation (CV) values of 14% (injection replica) and 32% (reaction replica). Thus our results demonstrate that the reproducibility of QCLMS is in line with the reproducibility of general quantitative proteomics and we establish a robust workflow for MS1-based quantitation of cross-linked peptides.

Quantitative proteomics analysis reveals perturbation of lipid metabolic pathways in the liver of Atlantic cod (Gadus morhua) treated with PCB 153.

Science.gov (United States)

Yadetie, Fekadu; Oveland, Eystein; Døskeland, Anne; Berven, Frode; Goksøyr, Anders; Karlsen, Odd André

2017-04-01

PCB 153 is one of the most abundant PCB congeners detected in biological samples. It is a persistent compound that is still present in the environment despite the ban on production and use of PCBs in the late 1970s. It has strong tendencies to bioaccumulate and biomagnify in biota, and studies have suggested that it is an endocrine and metabolic disruptor. In order to study mechanisms of toxicity, we exposed Atlantic cod (Gadus morhua) to various doses of PCB 153 (0, 0.5, 2 and 8mg/kg body weight) for two weeks and examined the effects on expression of liver proteins using label-free quantitative proteomics. Label-free liquid chromatography-mass spectrometry analysis of the liver proteome resulted in the quantification of 1272 proteins, of which 78 proteins were differentially regulated in the PCB 153-treated dose groups compared to the control group. Functional enrichment analysis showed that pathways significantly affected are related to lipid metabolism, cytoskeletal remodeling, cell cycle and cell adhesion. Importantly, the main effects appear to be on lipid metabolism, with up-regulation of enzymes in the de novo fatty acid synthesis pathway, consistent with previous transcriptomics results. Increased plasma triglyceride levels were also observed in the PCB 153 treated fish, in agreement with the induction of the lipogenic genes and proteins. The results suggest that PCB 153 perturbs lipid metabolism in the Atlantic cod liver. Elevated levels of lipogenic enzymes and plasma triglycerides further suggest increased synthesis of fatty acids and triglycerides. Copyright © 2017 Elsevier B.V. All rights reserved.

Relative Quantitative Proteomic Analysis of Brucella abortus Reveals Metabolic Adaptation to Multiple Environmental Stresses

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Xiaodong Zai

2017-11-01

Full Text Available Brucella spp. are facultative intracellular pathogens that cause chronic brucellosis in humans and animals. The virulence of Brucella primarily depends on its successful survival and replication in host cells. During invasion of the host tissue, Brucella is simultaneously subjected to a variety of harsh conditions, including nutrient limitation, low pH, antimicrobial defenses, and extreme levels of reactive oxygen species (ROS via the host immune response. This suggests that Brucella may be able to regulate its metabolic adaptation in response to the distinct stresses encountered during its intracellular infection of the host. An investigation into the differential proteome expression patterns of Brucella grown under the relevant stress conditions may contribute toward a better understanding of its pathogenesis and adaptive response. Here, we utilized a mass spectrometry-based label-free relative quantitative proteomics approach to investigate and compare global proteomic changes in B. abortus in response to eight different stress treatments. The 3 h short-term in vitro single-stress and multi-stress conditions mimicked the in vivo conditions of B. abortus under intracellular infection, with survival rates ranging from 3.17 to 73.17%. The proteomic analysis identified and quantified a total of 2,272 proteins and 74% of the theoretical proteome, thereby providing wide coverage of the B. abortus proteome. By including eight distinct growth conditions and comparing these with a control condition, we identified a total of 1,221 differentially expressed proteins (DEPs that were significantly changed under the stress treatments. Pathway analysis revealed that most of the proteins were involved in oxidative phosphorylation, ABC transporters, two-component systems, biosynthesis of secondary metabolites, the citrate cycle, thiamine metabolism, and nitrogen metabolism; constituting major response mechanisms toward the reconstruction of cellular

Characterization of human myotubes from type 2 diabetic and non-diabetic subjects using complementary quantitative mass spectrometric methods

DEFF Research Database (Denmark)

Thingholm, Tine E; Bak, Steffen; Beck-Nielsen, Henning

2011-01-01

2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery...... strategy using iTRAQ and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring (SRM) to identify, quantify and validate changes in protein abundance between human myotubes obtained from non-diabetic lean, non-diabetic obese and type 2 diabetic subjects...

A comparative study of fat storage quantitation in nematode Caenorhabditis elegans using label and label-free methods.

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Kelvin Yen

to the staining of fat stores, but rather the sequestration of dyes in lysosome-related organelles. In contrast, fixative staining methods provide reproducible data but are prone to errors due to the interference of autofluorescent species and the non-specific staining of cellular structures other than fat stores. Importantly, both growth conditions and developmental stage should be considered when comparing methods of C. elegans lipid storage. Taken together, we confirm that CARS microscopy provides a direct, non-invasive, and label-free means to quantitatively analyze fat storage in living C. elegans.

Development and standardization of multiplexed antibody microarrays for use in quantitative proteomics

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Sorette M

2004-12-01

Full Text Available Abstract Background Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification. Results Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies. Conclusion The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings.

Quantitative proteomic assessment of very early cellular signaling events

DEFF Research Database (Denmark)

Dengjel, Joern; Akimov, Vyacheslav; Olsen, Jesper V

2007-01-01

Technical limitations have prevented proteomic analyses of events occurring less than 30 s after signal initiation. We developed an automated, continuous quench-flow system allowing quantitative proteomic assessment of very early cellular signaling events (qPACE) with a time resolution of 1 s...

Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

Energy Technology Data Exchange (ETDEWEB)

Liu, Xing [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Xu, Yanli [Fuyang People’s Hospital (China); Meng, Qian [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Zheng, Qingqing [Digestive Endoscopic Center, Shanghai Jiaotong University Affiliated Sixth People’s Hospital (China); Wu, Jianhong [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China); Wang, Chen; Jia, Weiping [Shanghai Key Laboratory of Diabetes Mellitus, Department of Endocrinology and Metabolism, Shanghai Diabetes Institute, Shanghai Clinical Center for Diabetes, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital (China); Figeys, Daniel [Department of Biochemistry, Microbiology and Immunology, and Department of Chemistry and Biomolecular Sciences, University of Ottawa (Canada); Chang, Ying, E-mail: emulan@163.com [Digestive Endoscopic Center, Shanghai Jiaotong University Affiliated Sixth People’s Hospital (China); Zhou, Hu, E-mail: zhouhu@simm.ac.cn [Department of Analytical Chemistry and CAS Key Laboratory of Receptor Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences (China)

2016-08-05

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.

Proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling

International Nuclear Information System (INIS)

Liu, Xing; Xu, Yanli; Meng, Qian; Zheng, Qingqing; Wu, Jianhong; Wang, Chen; Jia, Weiping; Figeys, Daniel; Chang, Ying; Zhou, Hu

2016-01-01

Colorectal cancer (CRC) is one of the most common types of malignant tumor worldwide. Currently, although many researchers have been devoting themselves in CRC studies, the process of locating biomarkers for CRC early diagnosis and prognostic is still very slow. Using a centrifugal proteomic reactor-based proteomic analysis of minute amount of colonic biopsies by enteroscopy sampling, 2620 protein groups were quantified between cancer mucosa and adjacent normal colorectal mucosa. Of which, 403 protein groups were differentially expressed with statistic significance between cancer and normal tissues, including 195 up-regulated and 208 down-regulated proteins in cancer tissues. Three proteins (SOD3, PRELP and NGAL) were selected for further Western blot validation. And the resulting Western blot experimental results were consistent with the quantitative proteomic data. SOD3 and PRELP are down-regulated in CRC mucosa comparing to adjacent normal tissue, while NGAL is up-regulated in CRC mucosa. In conclusion, the centrifugal proteomic reactor-based label-free quantitative proteomic approach provides a highly sensitive and powerful tool for analyzing minute protein sample from tiny colorectal biopsies, which may facilitate CRC biomarkers discovery for diagnoses and prognoses. -- Highlights: •Minute amount of colonic biopsies by endoscopy is suitable for proteomic analysis. •Centrifugal proteomic reactor can be used for processing tiny clinic biopsy sample. •SOD3 and PRELP are down-regulated in CRC, while NGAL is up-regulated in CRC.

Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p.

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Virginie Redeker

Full Text Available BACKGROUND: The aggregation of the baker's yeast prion Sup35p is at the origin of the transmissible [PSI(+] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI(+] formation. RESULTS: We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI(+] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI(+] and [psi(-] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes. CONCLUSION: Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI(+] formation. Our results highlight the complexity of the cellular changes accompanying [PSI(+] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.

PIQMIe: A web server for semi-quantitative proteomics data management and analysis

NARCIS (Netherlands)

A. Kuzniar (Arnold); R. Kanaar (Roland)

2014-01-01

textabstractWe present the Proteomics Identifications and Quantitations Data Management and Integration Service or PIQMIe that aids in reliable and scalable data management, analysis and visualization of semi-quantitative mass spectrometry based proteomics experiments. PIQMIe readily integrates

Rapid label-free profiling of oral cancer biomarker proteins using nano-UPLC-Q-TOF ion mobility mass spectrometry.

Science.gov (United States)

Nassar, Ala F; Williams, Brad J; Yaworksy, Dustin C; Patel, Vyomesh; Rusling, James F

2016-03-01

It has become quite clear that single cancer biomarkers cannot in general provide high sensitivity and specificity for reliable clinical cancer diagnostics. This paper explores the feasibility of rapid detection of multiple biomarker proteins in model oral cancer samples using label-free protein relative quantitation. MS-based label-free quantitative proteomics offer a rapid alternative that bypasses the need for stable isotope containing compounds to chemically bind and label proteins. Total protein content in oral cancer cell culture conditioned media was precipitated, subjected to proteolytic digestion, and then analyzed using a nano-UPLC (where UPLC is ultra-performance liquid chromatography) coupled to a hybrid Q-Tof ion-mobility mass spectrometry (MS). Rapid, simultaneous identification and quantification of multiple possible cancer biomarker proteins was achieved. In a comparative study between cancer and noncancer samples, approximately 952 proteins were identified using a high-throughput 1D ion mobility assisted data independent acquisition (IM-DIA) approach. As we previously demonstrated that interleukin-8 (IL-8) and vascular endothelial growth factor A (VEGF-A) were readily detected in oral cancer cell conditioned media(1), we targeted these biomarker proteins to validate our approach. Target biomarker protein IL-8 was found between 3.5 and 8.8 fmol, while VEGF-A was found at 1.45 fmol in the cancer cell media. Overall, our data suggest that the nano-UPLC-IM-DIA bioassay is a feasible approach to identify and quantify proteins in complex samples without the need for stable isotope labeling. These results have significant implications for rapid tumor diagnostics and prognostics by monitoring proteins such as IL-8 and VEGF-A implicated in cancer development and progression. The analysis in tissue or plasma is not possible at this time, but the subsequent work would be needed for validation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative and temporal proteome analysis of butyrate-treated colorectal cancer cells.

Science.gov (United States)

Tan, Hwee Tong; Tan, Sandra; Lin, Qingsong; Lim, Teck Kwang; Hew, Choy Leong; Chung, Maxey C M

2008-06-01

Colorectal cancer is one of the most common cancers in developed countries, and its incidence is negatively associated with high dietary fiber intake. Butyrate, a short-chain fatty acid fermentation by-product of fiber induces cell maturation with the promotion of growth arrest, differentiation, and/or apoptosis of cancer cells. The stimulation of cell maturation by butyrate in colonic cancer cells follows a temporal progression from the early phase of growth arrest to the activation of apoptotic cascades. Previously we performed two-dimensional DIGE to identify differentially expressed proteins induced by 24-h butyrate treatment of HCT-116 colorectal cancer cells. Herein we used quantitative proteomics approaches using iTRAQ (isobaric tags for relative and absolute quantitation), a stable isotope labeling methodology that enables multiplexing of four samples, for a temporal study of HCT-116 cells treated with butyrate. In addition, cleavable ICAT, which selectively tags cysteine-containing proteins, was also used, and the results complemented those obtained from the iTRAQ strategy. Selected protein targets were validated by real time PCR and Western blotting. A model is proposed to illustrate our findings from this temporal analysis of the butyrate-responsive proteome that uncovered several integrated cellular processes and pathways involved in growth arrest, apoptosis, and metastasis. These signature clusters of butyrate-regulated pathways are potential targets for novel chemopreventive and therapeutic drugs for treatment of colorectal cancer.

Epsilon-Q: An Automated Analyzer Interface for Mass Spectral Library Search and Label-Free Protein Quantification.

Science.gov (United States)

Cho, Jin-Young; Lee, Hyoung-Joo; Jeong, Seul-Ki; Paik, Young-Ki

2017-12-01

Mass spectrometry (MS) is a widely used proteome analysis tool for biomedical science. In an MS-based bottom-up proteomic approach to protein identification, sequence database (DB) searching has been routinely used because of its simplicity and convenience. However, searching a sequence DB with multiple variable modification options can increase processing time, false-positive errors in large and complicated MS data sets. Spectral library searching is an alternative solution, avoiding the limitations of sequence DB searching and allowing the detection of more peptides with high sensitivity. Unfortunately, this technique has less proteome coverage, resulting in limitations in the detection of novel and whole peptide sequences in biological samples. To solve these problems, we previously developed the "Combo-Spec Search" method, which uses manually multiple references and simulated spectral library searching to analyze whole proteomes in a biological sample. In this study, we have developed a new analytical interface tool called "Epsilon-Q" to enhance the functions of both the Combo-Spec Search method and label-free protein quantification. Epsilon-Q performs automatically multiple spectral library searching, class-specific false-discovery rate control, and result integration. It has a user-friendly graphical interface and demonstrates good performance in identifying and quantifying proteins by supporting standard MS data formats and spectrum-to-spectrum matching powered by SpectraST. Furthermore, when the Epsilon-Q interface is combined with the Combo-Spec search method, called the Epsilon-Q system, it shows a synergistic function by outperforming other sequence DB search engines for identifying and quantifying low-abundance proteins in biological samples. The Epsilon-Q system can be a versatile tool for comparative proteome analysis based on multiple spectral libraries and label-free quantification.

A label-free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC-Chip coupled with Ion Trap mass spectrometry.

Science.gov (United States)

Brambilla, Francesca; Resta, Donatella; Isak, Ilena; Zanotti, Marco; Arnoldi, Anna

2009-01-01

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label-free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label-free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC-Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (beta-conglutin precursor and vicilin-like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin-encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the "normalized protein average of common reproducible peptides" (N-ACRP) for gamma-conglutin, which is a homogeneous protein, and the "normalized protein mean peptide spectral intensity" (N-MEAN) for the highly heterogenous class of the vicilins.

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples

Directory of Open Access Journals (Sweden)

Rígel Licier

2016-10-01

Full Text Available The proper handling of samples to be analyzed by mass spectrometry (MS can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples.

Science.gov (United States)

Licier, Rígel; Miranda, Eric; Serrano, Horacio

2016-10-17

The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

SAFER, an Analysis Method of Quantitative Proteomic Data, Reveals New Interactors of the C. elegans Autophagic Protein LGG-1.

Science.gov (United States)

Yi, Zhou; Manil-Ségalen, Marion; Sago, Laila; Glatigny, Annie; Redeker, Virginie; Legouis, Renaud; Mucchielli-Giorgi, Marie-Hélène

2016-05-06

Affinity purifications followed by mass spectrometric analysis are used to identify protein-protein interactions. Because quantitative proteomic data are noisy, it is necessary to develop statistical methods to eliminate false-positives and identify true partners. We present here a novel approach for filtering false interactors, named "SAFER" for mass Spectrometry data Analysis by Filtering of Experimental Replicates, which is based on the reproducibility of the replicates and the fold-change of the protein intensities between bait and control. To identify regulators or targets of autophagy, we characterized the interactors of LGG1, a ubiquitin-like protein involved in autophagosome formation in C. elegans. LGG-1 partners were purified by affinity, analyzed by nanoLC-MS/MS mass spectrometry, and quantified by a label-free proteomic approach based on the mass spectrometric signal intensity of peptide precursor ions. Because the selection of confident interactions depends on the method used for statistical analysis, we compared SAFER with several statistical tests and different scoring algorithms on this set of data. We show that SAFER recovers high-confidence interactors that have been ignored by the other methods and identified new candidates involved in the autophagy process. We further validated our method on a public data set and conclude that SAFER notably improves the identification of protein interactors.

Label-Free (XIC) Quantification of Venom Procoagulant and Neurotoxin Expression in Related Australian Elapid Snakes Gives Insight into Venom Toxicity Evolution.

Science.gov (United States)

Skejic, Jure; Steer, David L; Dunstan, Nathan; Hodgson, Wayne C

2015-11-06

This study demonstrates a direct role of venom protein expression alteration in the evolution of snake venom toxicity. Avian skeletal muscle contractile response to exogenously administered acetylcholine is completely inhibited upon exposure to South Australian and largely preserved following exposure to Queensland eastern brown snake Pseudonaja textilis venom, indicating potent postsynaptic neurotoxicity of the former and lack thereof of the latter venom. Label-free quantitative proteomics reveals extremely large differences in the expression of postsynaptic three-finger α-neurotoxins in these venoms, explaining the difference in the muscle contractile response and suggesting that the type of toxicity induced by venom can be modified by altered expression of venom proteins. Furthermore, the onset of neuromuscular paralysis in the rat phrenic nerve-diaphragm preparation occurs sooner upon exposure to the venom (10 μg/mL) with high expression of α-neurotoxins than the venoms containing predominately presynaptic β-neurotoxins. The study also finds that the onset of rat plasma coagulation is faster following exposure to the venoms with higher expression of venom prothrombin activator subunits. This is the first quantitative proteomic study that uses extracted ion chromatogram peak areas (MS1 XIC) of distinct homologous tryptic peptides to directly show the differences in the expression of venom proteins.

Proteomic and Microscopic Strategies towards the Analysis of the Cytoskeletal Networks in Major Neuropsychiatric Disorders

Directory of Open Access Journals (Sweden)

Joëlle V. F. Coumans

2016-04-01

Full Text Available Mental health disorders have become worldwide health priorities. It is estimated that in the next 20 years they will account for a 16 trillion United State dollars (US$ loss. Up to now, the underlying pathophysiology of psychiatric disorders remains elusive. Altered cytoskeleton proteins expression that may influence the assembly, organization and maintenance of cytoskeletal integrity has been reported in major depressive disorders, schizophrenia and to some extent bipolar disorders. The use of quantitative proteomics, dynamic microscopy and super-resolution microscopy to investigate disease-specific protein signatures holds great promise to improve our understanding of these disorders. In this review, we present the currently available quantitative proteomic approaches use in neurology, gel-based, stable isotope-labelling and label-free methodologies and evaluate their strengths and limitations. We also reported on enrichment/subfractionation methods that target the cytoskeleton associated proteins and discuss the need of alternative methods for further characterization of the neurocytoskeletal proteome. Finally, we present live cell imaging approaches and emerging dynamic microscopy technology that will provide the tools necessary to investigate protein interactions and their dynamics in the whole cells. While these areas of research are still in their infancy, they offer huge potential towards the understanding of the neuronal network stability and its modification across neuropsychiatric disorders.

Artificial intelligence in label-free microscopy biological cell classification by time stretch

CERN Document Server

Mahjoubfar, Ata; Jalali, Bahram

2017-01-01

This book introduces time-stretch quantitative phase imaging (TS-QPI), a high-throughput label-free imaging flow cytometer developed for big data acquisition and analysis in phenotypic screening. TS-QPI is able to capture quantitative optical phase and intensity images simultaneously, enabling high-content cell analysis, cancer diagnostics, personalized genomics, and drug development. The authors also demonstrate a complete machine learning pipeline that performs optical phase measurement, image processing, feature extraction, and classification, enabling high-throughput quantitative imaging that achieves record high accuracy in label -free cellular phenotypic screening and opens up a new path to data-driven diagnosis. • Demonstrates how machine learning is used in high-speed microscopy imaging to facilitate medical diagnosis; • Provides a systematic and comprehensive illustration of time stretch technology; • Enables multidisciplinary application, including industrial, biomedical, and artificial intell...

Stable isotope labeling by amino acids in cell culture (SILAC) and quantitative comparison of the membrane proteomes of self-renewing and differentiating human embryonic stem cells

DEFF Research Database (Denmark)

Prokhorova, Tatyana A; Rigbolt, Kristoffer T G; Johansen, Pia T

2009-01-01

Stable isotope labeling by amino acids in cell culture (SILAC) is a powerful quantitative proteomics platform for comprehensive characterization of complex biological systems. However, the potential of SILAC-based approaches has not been fully utilized in human embryonic stem cell (hESC) research...... embryonic stem cell lines. Of the 811 identified membrane proteins, six displayed significantly higher expression levels in the undifferentiated state compared with differentiating cells. This group includes the established marker CD133/Prominin-1 as well as novel candidates for hESC surface markers......: Glypican-4, Neuroligin-4, ErbB2, receptor-type tyrosine-protein phosphatase zeta (PTPRZ), and Glycoprotein M6B. Our study also revealed 17 potential markers of hESC differentiation as their corresponding protein expression levels displayed a dramatic increase in differentiated embryonic stem cell...

A comprehensive proteomics study on platelet concentrates: Platelet proteome, storage time and Mirasol pathogen reduction technology.

Science.gov (United States)

Salunkhe, Vishal; De Cuyper, Iris M; Papadopoulos, Petros; van der Meer, Pieter F; Daal, Brunette B; Villa-Fajardo, María; de Korte, Dirk; van den Berg, Timo K; Gutiérrez, Laura

2018-03-19

Platelet concentrates (PCs) represent a blood transfusion product with a major concern for safety as their storage temperature (20-24°C) allows bacterial growth, and their maximum storage time period (less than a week) precludes complete microbiological testing. Pathogen inactivation technologies (PITs) provide an additional layer of safety to the blood transfusion products from known and unknown pathogens such as bacteria, viruses, and parasites. In this context, PITs, such as Mirasol Pathogen Reduction Technology (PRT), have been developed and are implemented in many countries. However, several studies have shown in vitro that Mirasol PRT induces a certain level of platelet shape change, hyperactivation, basal degranulation, and increased oxidative damage during storage. It has been suggested that Mirasol PRT might accelerate what has been described as the platelet storage lesion (PSL), but supportive molecular signatures have not been obtained. We aimed at dissecting the influence of both variables, that is, Mirasol PRT and storage time, at the proteome level. We present comprehensive proteomics data analysis of Control PCs and PCs treated with Mirasol PRT at storage days 1, 2, 6, and 8. Our workflow was set to perform proteomics analysis using a gel-free and label-free quantification (LFQ) approach. Semi-quantification was based on LFQ signal intensities of identified proteins using MaxQuant/Perseus software platform. Data are available via ProteomeXchange with identifier PXD008119. We identified marginal differences between Mirasol PRT and Control PCs during storage. However, those significant changes at the proteome level were specifically related to the functional aspects previously described to affect platelets upon Mirasol PRT. In addition, the effect of Mirasol PRT on the platelet proteome appeared not to be exclusively due to an accelerated or enhanced PSL. In summary, semi-quantitative proteomics allows to discern between proteome changes due to

Quantitative Analysis of Differential Proteome Expression in Bladder Cancer vs. Normal Bladder Cells Using SILAC Method.

Directory of Open Access Journals (Sweden)

Ganglong Yang

Full Text Available The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia, KK47 (low grade nonmuscle invasive bladder cancer, NMIBC, and YTS1 (metastatic bladder cancer have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.

Quantitative targeted proteomics for understanding the blood-brain barrier: towards pharmacoproteomics.

Science.gov (United States)

Ohtsuki, Sumio; Hirayama, Mio; Ito, Shingo; Uchida, Yasuo; Tachikawa, Masanori; Terasaki, Tetsuya

2014-06-01

The blood-brain barrier (BBB) is formed by brain capillary endothelial cells linked together via complex tight junctions, and serves to prevent entry of drugs into the brain. Multiple transporters are expressed at the BBB, where they control exchange of materials between the circulating blood and brain interstitial fluid, thereby supporting and protecting the CNS. An understanding of the BBB is necessary for efficient development of CNS-acting drugs and to identify potential drug targets for treatment of CNS diseases. Quantitative targeted proteomics can provide detailed information on protein expression levels at the BBB. The present review highlights the latest applications of quantitative targeted proteomics in BBB research, specifically to evaluate species and in vivo-in vitro differences, and to reconstruct in vivo transport activity. Such a BBB quantitative proteomics approach can be considered as pharmacoproteomics.

ExSTA: External Standard Addition Method for Accurate High-Throughput Quantitation in Targeted Proteomics Experiments.

Science.gov (United States)

Mohammed, Yassene; Pan, Jingxi; Zhang, Suping; Han, Jun; Borchers, Christoph H

2018-03-01

Targeted proteomics using MRM with stable-isotope-labeled internal-standard (SIS) peptides is the current method of choice for protein quantitation in complex biological matrices. Better quantitation can be achieved with the internal standard-addition method, where successive increments of synthesized natural form (NAT) of the endogenous analyte are added to each sample, a response curve is generated, and the endogenous concentration is determined at the x-intercept. Internal NAT-addition, however, requires multiple analyses of each sample, resulting in increased sample consumption and analysis time. To compare the following three methods, an MRM assay for 34 high-to-moderate abundance human plasma proteins is used: classical internal SIS-addition, internal NAT-addition, and external NAT-addition-generated in buffer using NAT and SIS peptides. Using endogenous-free chicken plasma, the accuracy is also evaluated. The internal NAT-addition outperforms the other two in precision and accuracy. However, the curves derived by internal vs. external NAT-addition differ by only ≈3.8% in slope, providing comparable accuracies and precision with good CV values. While the internal NAT-addition method may be "ideal", this new external NAT-addition can be used to determine the concentration of high-to-moderate abundance endogenous plasma proteins, providing a robust and cost-effective alternative for clinical analyses or other high-throughput applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative Proteomic Profiling of Mycobacterium bovis and BCG Vaccine Strains

KAUST Repository

Gao, Ge

2013-01-01

, 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

GProX, a user-friendly platform for bioinformatics analysis and visualization of quantitative proteomics data.

Science.gov (United States)

Rigbolt, Kristoffer T G; Vanselow, Jens T; Blagoev, Blagoy

2011-08-01

Recent technological advances have made it possible to identify and quantify thousands of proteins in a single proteomics experiment. As a result of these developments, the analysis of data has become the bottleneck of proteomics experiment. To provide the proteomics community with a user-friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)(1). The program requires no special bioinformatics training, as all functions of GProX are accessible within its graphical user-friendly interface which will be intuitive to most users. Basic features facilitate the uncomplicated management and organization of large data sets and complex experimental setups as well as the inspection and graphical plotting of quantitative data. These are complemented by readily available high-level analysis options such as database querying, clustering based on abundance ratios, feature enrichment tests for e.g. GO terms and pathway analysis tools. A number of plotting options for visualization of quantitative proteomics data is available and most analysis functions in GProX create customizable high quality graphical displays in both vector and bitmap formats. The generic import requirements allow data originating from essentially all mass spectrometry platforms, quantitation strategies and software to be analyzed in the program. GProX represents a powerful approach to proteomics data analysis providing proteomics experimenters with a toolbox for bioinformatics analysis of quantitative proteomics data. The program is released as open-source and can be freely downloaded from the project webpage at http://gprox.sourceforge.net.

Quantitative proteomic analysis of cabernet sauvignon grape cells exposed to thermal stresses reveals alterations in sugar and phenylpropanoid metabolism.

Science.gov (United States)

George, Iniga S; Pascovici, Dana; Mirzaei, Mehdi; Haynes, Paul A

2015-09-01

Grapes (Vitis vinifera) are a valuable fruit crop and wine production is a major industry. Global warming and expanded range of cultivation will expose grapes to more temperature stresses in future. Our study investigated protein level responses to abiotic stresses, with particular reference to proteomic changes induced by the impact of four different temperature stress regimes, including both hot and cold temperatures, on cultured grape cells. Cabernet Sauvignon cell suspension cultures grown at 26°C were subjected to 14 h of exposure to 34 and 42°C for heat stress, and 18 and 10°C for cold stress. Cells from the five temperatures were harvested in biological triplicates and label-free quantitative shotgun proteomic analysis was performed. A total of 2042 non-redundant proteins were identified from the five temperature points. Fifty-five proteins were only detected in extreme heat stress conditions (42°C) and 53 proteins were only detected at extreme cold stress conditions (10°C). Gene Ontology (GO) annotations of differentially expressed proteins provided insights into the metabolic pathways that are involved in temperature stress in grape cells. Sugar metabolism displayed switching between alternative and classical pathways during temperature stresses. Additionally, nine proteins involved in the phenylpropanoid pathway were greatly increased in abundance at extreme cold stress, and were thus found to be cold-responsive proteins. All MS data have been deposited in the ProteomeXchange with identifier PXD000977 (http://proteomecentral.proteomexchange.org/dataset/PXD000977). © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomic analysis of post-translational modifications of human histones

DEFF Research Database (Denmark)

Beck, Hans Christian; Nielsen, Eva C; Matthiesen, Rune

2006-01-01

, and H4 in a site-specific and dose-dependent manner. This unbiased analysis revealed that a relative increase in acetylated peptide from the histone variants H2A, H2B, and H4 was accompanied by a relative decrease of dimethylated Lys(57) from histone H2B. The dose-response results obtained...... by quantitative proteomics of histones from HDACi-treated cells were consistent with Western blot analysis of histone acetylation, cytotoxicity, and dose-dependent expression profiles of p21 and cyclin A2. This demonstrates that mass spectrometry-based quantitative proteomic analysis of post-translational...

Evaluation of Rice Resistance to Southern Rice Black-Streaked Dwarf Virus and Rice Ragged Stunt Virus through Combined Field Tests, Quantitative Real-Time PCR, and Proteome Analysis.

Science.gov (United States)

Wang, Zhenchao; Yu, Lu; Jin, Linhong; Wang, Wenli; Zhao, Qi; Ran, Longlu; Li, Xiangyang; Chen, Zhuo; Guo, Rong; Wei, Yongtian; Yang, Zhongcheng; Liu, Enlong; Hu, Deyu; Song, Baoan

2017-02-22

Diseases caused by southern rice black-streaked dwarf virus (SRBSDV) and rice ragged stunt virus (RRSV) considerably decrease grain yield. Therefore, determining rice cultivars with high resistance to SRBSDV and RRSV is necessary. In this study, rice cultivars with high resistance to SRBSDV and RRSV were evaluated through field trials in Shidian and Mangshi county, Yunnan province, China. SYBR Green I-based quantitative real-time polymerase chain reaction (qRT-PCR) analysis was used to quantitatively detect virus gene expression levels in different rice varieties. The following parameters were applied to evaluate rice resistance: acre yield (A.Y.), incidence of infected plants (I.I.P.), virus load (V.L.), disease index (D.I.), and insect quantity (I.Q.) per 100 clusters. Zhongzheyou1 (Z1) and Liangyou2186 (L2186) were considered the most suitable varieties with integrated higher A.Y., lower I.I.P., V.L., D.I. and I.Q. In order to investigate the mechanism of rice resistance, comparative label-free shotgun liquid chromatography tandem-mass spectrometry (LC-MS/MS) proteomic approaches were applied to comprehensively describe the proteomics of rice varieties' SRBSDV tolerance. Systemic acquired resistance (SAR)-related proteins in Z1 and L2186 may result in the superior resistance of these varieties compared with Fengyouxiangzhan (FYXZ).

Noninvasive diagnosis of intraamniotic infection: proteomic biomarkers in vaginal fluid.

Science.gov (United States)

Hitti, Jane; Lapidus, Jodi A; Lu, Xinfang; Reddy, Ashok P; Jacob, Thomas; Dasari, Surendra; Eschenbach, David A; Gravett, Michael G; Nagalla, Srinivasa R

2010-07-01

We analyzed the vaginal fluid proteome to identify biomarkers of intraamniotic infection among women in preterm labor. Proteome analysis was performed on vaginal fluid specimens from women with preterm labor, using multidimensional liquid chromatography, tandem mass spectrometry, and label-free quantification. Enzyme immunoassays were used to quantify candidate proteins. Classification accuracy for intraamniotic infection (positive amniotic fluid bacterial culture and/or interleukin-6 >2 ng/mL) was evaluated using receiver-operator characteristic curves obtained by logistic regression. Of 170 subjects, 30 (18%) had intraamniotic infection. Vaginal fluid proteome analysis revealed 338 unique proteins. Label-free quantification identified 15 proteins differentially expressed in intraamniotic infection, including acute-phase reactants, immune modulators, high-abundance amniotic fluid proteins and extracellular matrix-signaling factors; these findings were confirmed by enzyme immunoassay. A multi-analyte algorithm showed accurate classification of intraamniotic infection. Vaginal fluid proteome analyses identified proteins capable of discriminating between patients with and without intraamniotic infection. Copyright (c) 2010 Mosby, Inc. All rights reserved.

GProX, a User-Friendly Platform for Bioinformatics Analysis and Visualization of Quantitative Proteomics Data

DEFF Research Database (Denmark)

Rigbolt, Kristoffer T G; Vanselow, Jens T; Blagoev, Blagoy

2011-01-01

-friendly platform for comprehensive analysis, inspection and visualization of quantitative proteomics data we developed the Graphical Proteomics Data Explorer (GProX)(1). The program requires no special bioinformatics training, as all functions of GProX are accessible within its graphical user-friendly interface...... such as database querying, clustering based on abundance ratios, feature enrichment tests for e.g. GO terms and pathway analysis tools. A number of plotting options for visualization of quantitative proteomics data is available and most analysis functions in GProX create customizable high quality graphical...... displays in both vector and bitmap formats. The generic import requirements allow data originating from essentially all mass spectrometry platforms, quantitation strategies and software to be analyzed in the program. GProX represents a powerful approach to proteomics data analysis providing proteomics...

Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity.

Science.gov (United States)

Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Fallon, John K; Barber, Jill; Smith, Philip C; Rostami-Hodjegan, Amin; Goosen, Theunis C

2017-10-01

Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes ( n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Quantitative and qualitative proteome characteristics extracted from in-depth integrated genomics and proteomics analysis

NARCIS (Netherlands)

Low, T.Y.; van Heesch, S.; van den Toorn, H.; Giansanti, P.; Cristobal, A.; Toonen, P.; Schafer, S.; Hubner, N.; van Breukelen, B.; Mohammed, S.; Cuppen, E.; Heck, A.J.R.; Guryev, V.

2013-01-01

Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and posttranscriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We

Proteomic analysis of bronchoalveolar lavage fluid (BALF) from lung cancer patients using label-free mass spectrometry.

Science.gov (United States)

Hmmier, Abduladim; O'Brien, Michael Emmet; Lynch, Vincent; Clynes, Martin; Morgan, Ross; Dowling, Paul

2017-06-01

Lung cancer is the leading cause of cancer-related mortality in both men and women throughout the world. The need to detect lung cancer at an early, potentially curable stage, is essential and may reduce mortality by 20%. The aim of this study was to identify distinct proteomic profiles in bronchoalveolar fluid (BALF) and plasma that are able to discriminate individuals with benign disease from those with non-small cell lung cancer (NSCLC). Using label-free mass spectrometry analysis of BALF during discovery-phase analysis, a significant number of proteins were found to have different abundance levels when comparing control to adenocarcinoma (AD) or squamous cell lung carcinoma (SqCC). Validation of candidate biomarkers identified in BALF was performed in a larger cohort of plasma samples by detection with enzyme-linked immunoassay. Four proteins (Cystatin-C, TIMP-1, Lipocalin-2 and HSP70/HSPA1A) were selected as a representative group from discovery phase mass spectrometry BALF analysis. Plasma levels of TIMP-1, Lipocalin-2 and Cystatin-C were found to be significantly elevated in AD and SqCC compared to control. The results presented in this study indicate that BALF is an important proximal biofluid for the discovery and identification of candidate lung cancer biomarkers. There is good correlation between the trend of protein abundance levels in BALF and that of plasma which validates this approach to develop a blood biomarker to aid lung cancer diagnosis, particularly in the era of lung cancer screening. The protein signatures identified also provide insight into the molecular mechanisms associated with lung malignancy.

Quantitative and Qualitative Proteome Characteristics Extracted from In-Depth Integrated Genomics and Proteomics Analysis

NARCIS (Netherlands)

Low, Teck Yew; van Heesch, Sebastiaan; van den Toorn, Henk; Giansanti, Piero; Cristobal, Alba; Toonen, Pim; Schafer, Sebastian; Huebner, Norbert; van Breukelen, Bas; Mohammed, Shabaz; Cuppen, Edwin; Heck, Albert J. R.; Guryev, Victor

2013-01-01

Quantitative and qualitative protein characteristics are regulated at genomic, transcriptomic, and post-transcriptional levels. Here, we integrated in-depth transcriptome and proteome analyses of liver tissues from two rat strains to unravel the interactions within and between these layers. We

Insights from quantitative metaproteomics and protein-stable isotope probing into microbial ecology.

Science.gov (United States)

von Bergen, Martin; Jehmlich, Nico; Taubert, Martin; Vogt, Carsten; Bastida, Felipe; Herbst, Florian-Alexander; Schmidt, Frank; Richnow, Hans-Hermann; Seifert, Jana

2013-10-01

The recent development of metaproteomics has enabled the direct identification and quantification of expressed proteins from microbial communities in situ, without the need for microbial enrichment. This became possible by (1) significant increases in quality and quantity of metagenome data and by improvements of (2) accuracy and (3) sensitivity of modern mass spectrometers (MS). The identification of physiologically relevant enzymes can help to understand the role of specific species within a community or an ecological niche. Beside identification, relative and absolute quantitation is also crucial. We will review label-free and label-based methods of quantitation in MS-based proteome analysis and the contribution of quantitative proteome data to microbial ecology. Additionally, approaches of protein-based stable isotope probing (protein-SIP) for deciphering community structures are reviewed. Information on the species-specific metabolic activity can be obtained when substrates or nutrients are labeled with stable isotopes in a protein-SIP approach. The stable isotopes ((13)C, (15)N, (36)S) are incorporated into proteins and the rate of incorporation can be used for assessing the metabolic activity of the corresponding species. We will focus on the relevance of the metabolic and phylogenetic information retrieved with protein-SIP studies and for detecting and quantifying the carbon flux within microbial consortia. Furthermore, the combination of protein-SIP with established tools in microbial ecology such as other stable isotope probing techniques are discussed.

Molecular imaging of melanin distribution in vivo and quantitative differential diagnosis of human pigmented lesions using label-free harmonic generation biopsy (Conference Presentation)

Science.gov (United States)

Sun, Chi-Kuang; Wei, Ming-Liang; Su, Yu-Hsiang; Weng, Wei-Hung; Liao, Yi-Hua

2017-02-01

Harmonic generation microscopy is a noninvasive repetitive imaging technique that provides real-time 3D microscopic images of human skin with a sub-femtoliter resolution and high penetration down to the reticular dermis. In this talk, we show that with a strong resonance effect, the third-harmonic-generation (THG) modality provides enhanced contrast on melanin and allows not only differential diagnosis of various pigmented skin lesions but also quantitative imaging for longterm tracking. This unique capability makes THG microscopy the only label-free technique capable of identifying the active melanocytes in human skin and to image their different dendriticity patterns. In this talk, we will review our recent efforts to in vivo image melanin distribution and quantitatively diagnose pigmented skin lesions using label-free harmonic generation biopsy. This talk will first cover the spectroscopic study on the melanin enhanced THG effect in human cells and the calibration strategy inside human skin for quantitative imaging. We will then review our recent clinical trials including: differential diagnosis capability study on pigmented skin tumors; as well as quantitative virtual biopsy study on pre- and post- treatment evaluation on melasma and solar lentigo. Our study indicates the unmatched capability of harmonic generation microscopy to perform virtual biopsy for noninvasive histopathological diagnosis of various pigmented skin tumors, as well as its unsurpassed capability to noninvasively reveal the pathological origin of different hyperpigmentary diseases on human face as well as to monitor the efficacy of laser depigmentation treatments. This work is sponsored by National Health Research Institutes.

Quantitative profiling of serum samples using TMT protein labelling, fractionation and LC-MS/MS.

Science.gov (United States)

Sinclair, John; Timms, John F

2011-08-01

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.

Nucleophosmin in the pathogenesis of arsenic-related bladder carcinogenesis revealed by quantitative proteomics

International Nuclear Information System (INIS)

Chen Shuhui; Wang Yiwen; Hsu Jueliang; Chang Hongyi; Wang Chiyun; Shen Potsun; Chiang Chiwu; Chuang Jingjing; Tsai Hungwen; Gu Powen; Chang Fangchih; Liu Hsiaosheng; Chow Nanhaw

2010-01-01

To investigate the molecular mechanisms of arsenic (As)-associated carcinogenesis, we performed proteomic analysis on E7 immortalized human uroepithelial cells after treatment with As in vitro. Quantitative proteomics was performed using stable isotope dimethyl labeling coupled with two-dimensional liquid chromatography peptide separation and mass spectrometry (MS)/MS analysis. Among 285 proteins, a total of 26 proteins were upregulated (ratio > 2.0) and 18 proteins were downregulated (ratio < 0.65) by As treatment, which are related to nucleotide binding, lipid metabolism, protein folding, protein biosynthesis, transcription, DNA repair, cell cycle control, and signal transduction. This study reports the potential significance of nucleophosmin (NPM) in the As-related bladder carcinogenesis. NPM was universally expressed in all of uroepithelial cell lines examined, implying that NPM may play a role in human bladder carcinogenesis. Upregulation of NPM tends to be dose- and time-dependent after As treatment. Expression of NPM was associated with cell proliferation, migration and anti-apoptosis. On the contrary, soy isoflavones inhibited the expression of NPM in vitro. The results suggest that NPM may play a role in the As-related bladder carcinogenesis, and soybean-based foods may have potential in the suppression of As/NPM-related tumorigenesis.

Determination of variation parameters as a crucial step in designing TMT-based clinical proteomics experiments.

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Evelyne Maes

Full Text Available In quantitative shotgun proteomic analyses by liquid chromatography and mass spectrometry, a rigid study design is necessary in order to obtain statistically relevant results. Hypothesis testing, sample size calculation and power estimation are fundamental concepts that require consideration upon designing an experiment. For this reason, the reproducibility and variability of the proteomic platform needs to be assessed. In this study, we evaluate the technical (sample preparation, labeling (isobaric labels, and total (biological + technical + labeling + experimental variability and reproducibility of a workflow that employs a shotgun LC-MS/MS approach in combination with TMT peptide labeling for the quantification of peripheral blood mononuclear cell (PBMC proteome. We illustrate that the variability induced by TMT labeling is small when compared to the technical variation. The latter is also responsible for a substantial part of the total variation. Prior knowledge about the experimental variability allows for a correct design, a prerequisite for the detection of biologically significant disease-specific differential proteins in clinical proteomics experiments.

Use of label-free protein chips for proteomics of oncohematological diseases

Czech Academy of Sciences Publication Activity Database

Pimková, K.; Suttnar, J.; Bocková, Markéta; Homola, Jiří; Dyr, J. E.

2012-01-01

Roč. 58, SUPPL. 2 (2012), 2S84-2S89 ISSN 0042-773X Institutional support: RVO:67985882 Keywords : Oncohematological diseases * Surface plasmon resonance * Proteomics Subject RIV: JB - Sensors, Measurment, Regulation

Label and Label-Free Detection Techniques for Protein Microarrays

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Amir Syahir

2015-04-01

Full Text Available Protein microarray technology has gone through numerous innovative developments in recent decades. In this review, we focus on the development of protein detection methods embedded in the technology. Early microarrays utilized useful chromophores and versatile biochemical techniques dominated by high-throughput illumination. Recently, the realization of label-free techniques has been greatly advanced by the combination of knowledge in material sciences, computational design and nanofabrication. These rapidly advancing techniques aim to provide data without the intervention of label molecules. Here, we present a brief overview of this remarkable innovation from the perspectives of label and label-free techniques in transducing nano‑biological events.

MilQuant: a free, generic software tool for isobaric tagging-based quantitation.

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Zou, Xiao; Zhao, Minzhi; Shen, Hongyan; Zhao, Xuyang; Tong, Yuanpeng; Wang, Qingsong; Wei, Shicheng; Ji, Jianguo

2012-09-18

Isobaric tagging techniques such as iTRAQ and TMT are widely used in quantitative proteomics and especially useful for samples that demand in vitro labeling. Due to diversity in choices of MS acquisition approaches, identification algorithms, and relative abundance deduction strategies, researchers are faced with a plethora of possibilities when it comes to data analysis. However, the lack of generic and flexible software tool often makes it cumbersome for researchers to perform the analysis entirely as desired. In this paper, we present MilQuant, mzXML-based isobaric labeling quantitator, a pipeline of freely available programs that supports native acquisition files produced by all mass spectrometer types and collection approaches currently used in isobaric tagging based MS data collection. Moreover, aside from effective normalization and abundance ratio deduction algorithms, MilQuant exports various intermediate results along each step of the pipeline, making it easy for researchers to customize the analysis. The functionality of MilQuant was demonstrated by four distinct datasets from different laboratories. The compatibility and extendibility of MilQuant makes it a generic and flexible tool that can serve as a full solution to data analysis of isobaric tagging-based quantitation. Copyright © 2012 Elsevier B.V. All rights reserved.

Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?

Science.gov (United States)

Gizak, Agnieszka; Rakus, Dariusz

2016-01-11

Molecular and cellular biology methodology is traditionally based on the reasoning called "the mechanistic explanation". In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems' complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites), and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.

Will Quantitative Proteomics Redefine Some of the Key Concepts in Skeletal Muscle Physiology?

Directory of Open Access Journals (Sweden)

Agnieszka Gizak

2016-01-01

Full Text Available Molecular and cellular biology methodology is traditionally based on the reasoning called “the mechanistic explanation”. In practice, this means identifying and selecting correlations between biological processes which result from our manipulation of a biological system. In theory, a successful application of this approach requires precise knowledge about all parameters of a studied system. However, in practice, due to the systems’ complexity, this requirement is rarely, if ever, accomplished. Typically, it is limited to a quantitative or semi-quantitative measurements of selected parameters (e.g., concentrations of some metabolites, and a qualitative or semi-quantitative description of expression/post-translational modifications changes within selected proteins. A quantitative proteomics approach gives a possibility of quantitative characterization of the entire proteome of a biological system, in the context of the titer of proteins as well as their post-translational modifications. This enables not only more accurate testing of novel hypotheses but also provides tools that can be used to verify some of the most fundamental dogmas of modern biology. In this short review, we discuss some of the consequences of using quantitative proteomics to verify several key concepts in skeletal muscle physiology.

Quantitative Proteomic Analysis of Sulfolobus solfataricus Membrane Proteins

NARCIS (Netherlands)

Pham, T.K.; Sierocinski, P.; Oost, van der J.; Wright, P.C.

2010-01-01

A quantitative proteomic analysis of the membrane of the archaeon Sulfolobus solfataricus P2 using iTRAQ was successfully demonstrated in this technical note. The estimated number of membrane proteins of this organism is 883 (predicted based on Gravy score), corresponding to 30 % of the total

Scaffold-free, label-free and nozzle-free biofabrication technology using magnetic levitational assembly.

Science.gov (United States)

Parfenov, Vladislav A; Koudan, Elizaveta V; Bulanova, Elena A; Karalkin, Pavel A; Pereira, Frederico DAS; Norkin, Nikita E; Knyazeva, Alisa D; Gryadunova, Anna A; Petrov, Oleg F; Vasiliev, M M; Myasnikov, Maxim; Chernikov, Valery P; Kasyanov, Vladimir A; Marchenkov, Artem Yu; Brakke, Kenneth A; Khesuani, Yusef D; Demirci, Utkan; Mironov, Vladimir A

2018-05-31

Tissue spheroids have been proposed as building blocks in 3D biofabrication. Conventional magnetic force-driven 2D patterning of tissue spheroids requires prior cell labeling by magnetic nanoparticles, meanwhile a label-free approach for 3D magnetic levitational assembly has been introduced. Here we present first-time report on rapid assembly of 3D tissue construct using scaffold-free, nozzle-free and label-free magnetic levitation of tissue spheroids. Chondrospheres of standard size, shape and capable to fusion have been biofabricated from primary sheep chondrocytes using non-adhesive technology. Label-free magnetic levitation was performed using a prototype device equipped with permanent magnets in presence of gadolinium (Gd3+) in culture media, which enables magnetic levitation. Mathematical modeling and computer simulations were used for prediction of magnetic field and kinetics of tissue spheroids assembly into 3D tissue constructs. First, we used polystyrene beads to simulate the assembly of tissue spheroids and to determine the optimal settings for magnetic levitation in presence of Gd3+. Second, we proved the ability of chondrospheres to assemble rapidly into 3D tissue construct in the permanent magnetic field in the presence of Gd3+. Thus, scaffold- and label-free magnetic levitation of tissue spheroids is a promising approach for rapid 3D biofabrication and attractive alternative to label-based magnetic force-driven tissue engineering. . © 2018 IOP Publishing Ltd.

Label-Free Quantitative Proteomics of Embryogenic and Non-Embryogenic Callus during Sugarcane Somatic Embryogenesis.

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Angelo Schuabb Heringer

Full Text Available The development of somatic cells in to embryogenic cells occurs in several stages and ends in somatic embryo formation, though most of these biochemical and molecular changes have yet to be elucidated. Somatic embryogenesis coupled with genetic transformation could be a biotechnological tool to improve potential crop yields potential in sugarcane cultivars. The objective of this study was to observe somatic embryo development and to identify differentially expressed proteins in embryogenic (E and non-embryogenic (NE callus during maturation treatment. E and NE callus were cultured on maturation culture medium supplemented with different concentrations (0.0, 0.75, 1.5 and 2.0 g L(-1 of activated charcoal (AC. Somatic embryo formation and differential protein expression were evaluated at days 0 and 21 using shotgun proteomic analyses. Treatment with 1.5 g L(-1 AC resulted in higher somatic embryo maturation rates (158 somatic embryos in 14 days in E callus but has no effect in NE callus. A total of 752 co-expressed proteins were identified through the SUCEST (The Sugarcane EST Project, including many housekeeping proteins. E callus showed 65 exclusive proteins on day 0, including dehydrogenase, desiccation-related protein, callose synthase 1 and nitric oxide synthase. After 21 days on maturation treatment, 14 exclusive proteins were identified in E callus, including catalase and secreted protein. NE callus showed 23 exclusive proteins on day 0 and 10 exclusive proteins after 21 days on maturation treatment, including many proteins related to protein degradation. The induction of maturation leads to somatic embryo development, which likely depends on the expression of specific proteins throughout the process, as seen in E callus under maturation treatment. On the other hand, some exclusive proteins can also specifically prevent of somatic embryos development, as seen in the NE callus.

Multiplex N-terminome analysis of MMP-2 and MMP-9 substrate degradomes by iTRAQ-TAILS quantitative proteomics.

Science.gov (United States)

Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S; Overall, Christopher M

2010-05-01

Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses

Population-specific plasma proteomes of marine and freshwater three-spined sticklebacks (Gasterosteus aculeatus).

Science.gov (United States)

Kültz, Dietmar; Li, Johnathon; Zhang, Xuezhen; Villarreal, Fernando; Pham, Tuan; Paguio, Darlene

2015-12-01

Molecular phenotypes that distinguish resident marine (Bodega Harbor) from landlocked freshwater (FW, Lake Solano) three-spined sticklebacks were revealed by label-free quantitative proteomics. Secreted plasma proteins involved in lipid transport, blood coagulation, proteolysis, plasminogen-activating cascades, extracellular stimulus responses, and immunity are most abundant in this species. Globulins and albumins are much less abundant than in mammalian plasma. Unbiased quantitative proteome profiling identified 45 highly population-specific plasma proteins. Population-specific abundance differences were validated by targeted proteomics based on data-independent acquisition. Gene ontology enrichment analyses and known functions of population-specific plasma proteins indicate enrichment of processes controlling cell adhesion, tissue remodeling, proteolytic processing, and defense signaling in marine sticklebacks. Moreover, fetuin B and leukocyte cell derived chemotaxin 2 are much more abundant in marine fish. These proteins promote bone morphogenesis and likely contribute to population-specific body armor differences. Plasma proteins enriched in FW fish promote translation, heme biosynthesis, and lipid transport, suggesting a greater presence of plasma microparticles. Many prominent population-specific plasma proteins (e.g. apoptosis-associated speck-like protein containing a CARD) lack any homolog of known function or adequate functional characterization. Their functional characterization and the identification of population-specific environmental contexts and selective pressures that cause plasma proteome diversification are future directions emerging from this study. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Label-free quantitative mass spectrometry for analysis of protein antigens in a meningococcal group B outer membrane vesicle vaccine.

Science.gov (United States)

Dick, Lawrence W; Mehl, John T; Loughney, John W; Mach, Anna; Rustandi, Richard R; Ha, Sha; Zhang, Lan; Przysiecki, Craig T; Dieter, Lance; Hoang, Van M

2015-01-01

The development of a multivalent outer membrane vesicle (OMV) vaccine where each strain contributes multiple key protein antigens presents numerous analytical challenges. One major difficulty is the ability to accurately and specifically quantitate each antigen, especially during early development and process optimization when immunoreagents are limited or unavailable. To overcome this problem, quantitative mass spectrometry methods can be used. In place of traditional mass assays such as enzyme-linked immunosorbent assays (ELISAs), quantitative LC-MS/MS using multiple reaction monitoring (MRM) can be used during early-phase process development to measure key protein components in complex vaccines in the absence of specific immunoreagents. Multiplexed, label-free quantitative mass spectrometry methods using protein extraction by either detergent or 2-phase solvent were developed to quantitate levels of several meningococcal serogroup B protein antigens in an OMV vaccine candidate. Precision was demonstrated to be less than 15% RSD for the 2-phase extraction and less than 10% RSD for the detergent extraction method. Accuracy was 70 to 130% for the method using a 2-phase extraction and 90-110% for detergent extraction. The viability of MS-based protein quantification as a vaccine characterization method was demonstrated and advantages over traditional quantitative methods were evaluated. Implementation of these MS-based quantification methods can help to decrease the development time for complex vaccines and can provide orthogonal confirmation of results from existing antigen quantification techniques.

Science, marketing and wishful thinking in quantitative proteomics.

Science.gov (United States)

Hackett, Murray

2008-11-01

In a recent editorial (J. Proteome Res. 2007, 6, 1633) and elsewhere questions have been raised regarding the lack of attention paid to good analytical practice with respect to the reporting of quantitative results in proteomics. Using those comments as a starting point, several issues are discussed that relate to the challenges involved in achieving adequate sampling with MS-based methods in order to generate valid data for large-scale studies. The discussion touches on the relationships that connect sampling depth and the power to detect protein abundance change, conflict of interest, and strategies to overcome bureaucratic obstacles that impede the use of peer-to-peer technologies for transfer and storage of large data files generated in such experiments.

Quantitative and Label-Free Detection of Protein Kinase A Activity Based on Surface-Enhanced Raman Spectroscopy with Gold Nanostars.

Science.gov (United States)

He, Shuai; Kyaw, Yi Mon Ei; Tan, Eddie Khay Ming; Bekale, Laurent; Kang, Malvin Wei Cherng; Kim, Susana Soo-Yeon; Tan, Ivan; Lam, Kong-Peng; Kah, James Chen Yong

2018-04-26

The activity of extracellular protein kinase A (PKA) is known to be a biomarker for cancer. However, conventional PKA assays based on colorimetric, radioactive, and fluorometric techniques suffer from intensive labeling-related preparations, background interference, photobleaching, and safety concerns. While surface-enhanced Raman spectroscopy (SERS)-based assays have been developed for various enzymes to address these limitations, their use in probing PKA activity is limited due to subtle changes in the Raman spectrum with phosphorylation. Here, we developed a robust colloidal SERS-based scheme for label-free quantitative measurement of PKA activity using gold nanostars (AuNS) as a SERS substrate functionalized with bovine serum albumin (BSA)-kemptide (Kem) bioconjugate (AuNS-BSA-Kem), where BSA conferred colloidal stability and Kem is a high-affinity peptide substrate for PKA. By performing principle component analysis (PCA) on the SERS spectrum, we identified two Raman peaks at 725 and 1395 cm -1 , whose ratiometric intensity change provided a quantitative measure of Kem phosphorylation by PKA in vitro and allowed us to distinguish MDA-MB-231 and MCF-7 breast cancer cells known to overexpress extracellular PKA catalytic subunits from noncancerous human umbilical vein endothelial cells (HUVEC) based on their PKA activity in cell culture supernatant. The outcome demonstrated potential application of AuNS-BSA-Kem as a SERS probe for cancer screening based on PKA activity.

Phase sensitive spectral domain interferometry for label free biomolecular interaction analysis and biosensing applications

Science.gov (United States)

Chirvi, Sajal

-channel label-free biosensing applications is introduced. Simultaneous interrogation of multiple biosensors is achievable with a single spectral domain phase sensitive interferometer by coding the individual sensograms in coherence-multiplexed channels. Experimental results demonstrating multiplexed quantitative biomolecular interaction analysis of antibodies binding to antigen coated functionalized biosensor chip surfaces on different platforms are presented.

Statistical methods for quantitative mass spectrometry proteomic experiments with labeling

Directory of Open Access Journals (Sweden)

Oberg Ann L

2012-11-01

Full Text Available Abstract Mass Spectrometry utilizing labeling allows multiple specimens to be subjected to mass spectrometry simultaneously. As a result, between-experiment variability is reduced. Here we describe use of fundamental concepts of statistical experimental design in the labeling framework in order to minimize variability and avoid biases. We demonstrate how to export data in the format that is most efficient for statistical analysis. We demonstrate how to assess the need for normalization, perform normalization, and check whether it worked. We describe how to build a model explaining the observed values and test for differential protein abundance along with descriptive statistics and measures of reliability of the findings. Concepts are illustrated through the use of three case studies utilizing the iTRAQ 4-plex labeling protocol.

Statistical methods for quantitative mass spectrometry proteomic experiments with labeling.

Science.gov (United States)

Oberg, Ann L; Mahoney, Douglas W

2012-01-01

Mass Spectrometry utilizing labeling allows multiple specimens to be subjected to mass spectrometry simultaneously. As a result, between-experiment variability is reduced. Here we describe use of fundamental concepts of statistical experimental design in the labeling framework in order to minimize variability and avoid biases. We demonstrate how to export data in the format that is most efficient for statistical analysis. We demonstrate how to assess the need for normalization, perform normalization, and check whether it worked. We describe how to build a model explaining the observed values and test for differential protein abundance along with descriptive statistics and measures of reliability of the findings. Concepts are illustrated through the use of three case studies utilizing the iTRAQ 4-plex labeling protocol.

Non-biased enrichment does not improve quantitative proteomic delineation of reovirus T3D-infected HeLa cell protein alterations

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Jieyuan eJiang

2012-09-01

Full Text Available Mass spectrometry-based methods have allowed elucidation of alterations in complex proteomes, such as eukaryotic cells. Such studies have identified and measured relative abundances of thousands of host proteins after cells are infected with a virus. One of the potential limitations in such studies is that generally only the most abundant proteins are identified, leaving the deep richness of the cellular proteome largely unexplored. We differentially labeled HeLa cells with light and heavy stable isotopic forms of lysine and arginine (SILAC and infected cells with reovirus strain T3D. Cells were harvested at 24 hours post-infection. Heavy-labeled infected and light-labeled mock-infected cells were mixed together 1:1. Cells were then divided into cytosol and nuclear fractions and each fraction analyzed, both by standard 2D-HPLC/MS, and also after each fraction had been reacted with a random hexapeptide library (Proteominer® beads to attempt to enrich for low-abundance cellular proteins. A total of 2736 proteins were identified by 2 or more peptides at >99% confidence, of which 66 were significantly up-regulated and 67 were significantly down-regulated. Up-regulated proteins included those involved in antimicrobial and antiviral responses, GTPase activity, nucleotide binding, interferon signaling, and enzymes associated with energy generation. Down-regulated proteins included those involved in cell and biological adhesion, regulation of cell proliferation, structural molecule activity, and numerous molecular binding activities. Comparisons of the r2 correlations, degree of dataset overlap, and numbers of peptides detected suggest that non-biased enrichment approaches may not provide additional data to allow deeper quantitative and comparative mining of complex proteomes.

Quantitative proteome analysis reveals the correlation between endocytosis-associated proteins and hepatocellular carcinoma dedifferentiation.

Science.gov (United States)

Naboulsi, Wael; Bracht, Thilo; Megger, Dominik A; Reis, Henning; Ahrens, Maike; Turewicz, Michael; Eisenacher, Martin; Tautges, Stephanie; Canbay, Ali E; Meyer, Helmut E; Weber, Frank; Baba, Hideo A; Sitek, Barbara

2016-11-01

The majority of poorly differentiated hepatocellular carcinomas (HCCs) develop from well-differentiated tumors. Endocytosis is a cellular function which is likely to take part in this development due to its important role in regulating the abundances of vital signaling receptors. Here, we aimed to investigate the abundance of endocytosis-associated proteins in HCCs with various differentiation grades. Therefore, we analyzed 36 tissue specimens from HCC patients via LC-MS/MS-based label-free quantitative proteomics including 19 HCC tissue samples with different degrees of histological grades and corresponding non-tumorous tissue controls. As a result, 277 proteins were differentially regulated between well-differentiated tumors and controls. In moderately and poorly differentiated tumors, 278 and 1181 proteins, respectively, were significantly differentially regulated compared to non-tumorous tissue. We explored the regulated proteins based on their functions and identified thirty endocytosis-associated proteins, mostly overexpressed in poorly differentiated tumors. These included proteins that have been shown to be up-regulated in HCC like clathrin heavy chain-1 (CLTC) as well as unknown proteins, such as secretory carrier-associated membrane protein 3 (SCAMP3). The abundances of SCAMP3 and CLTC were immunohistochemically examined in tissue sections of 84 HCC patients. We demonstrate the novel association of several endocytosis-associated proteins, in particular, SCAMP3 with HCC progression. Copyright © 2016 Elsevier B.V. All rights reserved.

Application of mass spectrometry-based proteomics for biomarker discovery in neurological disorders

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Venugopal Abhilash

2009-01-01

Full Text Available Mass spectrometry-based quantitative proteomics has emerged as a powerful approach that has the potential to accelerate biomarker discovery, both for diagnostic as well as therapeutic purposes. Proteomics has traditionally been synonymous with 2D gels but is increasingly shifting to the use of gel-free systems and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS. Quantitative proteomic approaches have already been applied to investigate various neurological disorders, especially in the context of identifying biomarkers from cerebrospinal fluid and serum. This review highlights the scope of different applications of quantitative proteomics in understanding neurological disorders with special emphasis on biomarker discovery.

Proteomic analysis of mouse astrocytes and their secretome by a combination of FASP and StageTip-based, high pH, reversed-phase fractionation.

Science.gov (United States)

Han, Dohyun; Jin, Jonghwa; Woo, Jongmin; Min, Hophil; Kim, Youngsoo

2014-07-01

Astrocytes are the most abundant cells in the CNS, but their function remains largely unknown. Characterization of the whole-cell proteome and secretome in astrocytes would facilitate the study of their functions in various neurodegenerative diseases and astrocyte-neuron communication. To build a reference proteome, we established a C8-D1A astrocyte proteome to a depth of 7265 unique protein groups using a novel strategy that combined two-step digestion, filter-aided sample preparation, StageTip-based high pH fractionation, and high-resolution MS. Nearly, 6000 unique protein groups were identified from conditioned media of astrocyte cultures, constituting the largest astrocyte secretome that has been reported. High-confidence whole-cell proteomes and secretomes are valuable resources in studying astrocyte function by label-free quantitation and bioinformatics analysis. All MS data have been deposited in the ProteomeXchange with identifier PXD000501 (http://proteomecentral.proteomexchange.org/dataset/PXD000501). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Quantitative proteomics identify molecular targets that are crucial in larval settlement and metamorphosis of bugula neritina

KAUST Repository

Zhang, Huoming

2011-01-07

The marine invertebrate Bugula neritina has a biphasic life cycle that consists of a swimming larval stage and a sessile juvenile and adult stage. The attachment of larvae to the substratum and their subsequent metamorphosis have crucial ecological consequences. Despite many studies on this species, little is known about the molecular mechanism of these processes. Here, we report a comparative study of swimming larvae and metamorphosing individuals at 4 and 24 h postattachment using label-free quantitative proteomics. We identified more than 1100 proteins at each stage, 61 of which were differentially expressed. Specifically, proteins involved in energy metabolism and structural molecules were generally down-regulated, whereas proteins involved in transcription and translation, the extracellular matrix, and calcification were strongly up-regulated during metamorphosis. Many tightly regulated novel proteins were also identified. Subsequent analysis of the temporal and spatial expressions of some of the proteins and an assay of their functions indicated that they may have key roles in metamorphosis of B. neritina. These findings not only provide molecular evidence with which to elucidate the substantial changes in morphology and physiology that occur during larval attachment and metamorphosis but also identify potential targets for antifouling treatment. © 2011 American Chemical Society.

Real-Time, Label-Free Detection of Biomolecular Interactions in Sandwich Assays by the Oblique-Incidence Reflectivity Difference Technique

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Yung-Shin Sun

2014-12-01

Full Text Available One of the most important goals in proteomics is to detect the real-time kinetics of diverse biomolecular interactions. Fluorescence, which requires extrinsic tags, is a commonly and widely used method because of its high convenience and sensitivity. However, in order to maintain the conformational and functional integrality of biomolecules, label-free detection methods are highly under demand. We have developed the oblique-incidence reflectivity difference (OI-RD technique for label-free, kinetic measurements of protein-biomolecule interactions. Incorporating the total internal refection geometry into the OI-RD technique, we are able to detect as low as 0.1% of a protein monolayer, and this sensitivity is comparable with other label-free techniques such as surface plasmon resonance (SPR. The unique advantage of OI-RD over SPR is no need for dielectric layers. Moreover, using a photodiode array as the detector enables multi-channel detection and also eliminates the over-time signal drift. In this paper, we demonstrate the applicability and feasibility of the OI-RD technique by measuring the kinetics of protein-protein and protein-small molecule interactions in sandwich assays.

Identification of redox-sensitive cysteines in the arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method

KAUST Repository

Liu, Pei; Zhang, Huoming; Wang, Hai; Xia, Yiji

2014-01-01

-throughput quantitative proteomic approach termed OxiTRAQ for identifying proteins whose thiols undergo reversible oxidative modifications in Arabidopsis cells subjected to oxidative stress. In this approach, a biotinylated thiol-reactive reagent is used for differential

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

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Laëtitia Théron

2016-10-01

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.

Network analysis of quantitative proteomics on asthmatic bronchi: effects of inhaled glucocorticoid treatment

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Sihlbom Carina

2011-09-01

Full Text Available Abstract Background Proteomic studies of respiratory disorders have the potential to identify protein biomarkers for diagnosis and disease monitoring. Utilisation of sensitive quantitative proteomic methods creates opportunities to determine individual patient proteomes. The aim of the current study was to determine if quantitative proteomics of bronchial biopsies from asthmatics can distinguish relevant biological functions and whether inhaled glucocorticoid treatment affects these functions. Methods Endobronchial biopsies were taken from untreated asthmatic patients (n = 12 and healthy controls (n = 3. Asthmatic patients were randomised to double blind treatment with either placebo or budesonide (800 μg daily for 3 months and new biopsies were obtained. Proteins extracted from the biopsies were digested and analysed using isobaric tags for relative and absolute quantitation combined with a nanoLC-LTQ Orbitrap mass spectrometer. Spectra obtained were used to identify and quantify proteins. Pathways analysis was performed using Ingenuity Pathway Analysis to identify significant biological pathways in asthma and determine how the expression of these pathways was changed by treatment. Results More than 1800 proteins were identified and quantified in the bronchial biopsies of subjects. The pathway analysis revealed acute phase response signalling, cell-to-cell signalling and tissue development associations with proteins expressed in asthmatics compared to controls. The functions and pathways associated with placebo and budesonide treatment showed distinct differences, including the decreased association with acute phase proteins as a result of budesonide treatment compared to placebo. Conclusions Proteomic analysis of bronchial biopsy material can be used to identify and quantify proteins using highly sensitive technologies, without the need for pooling of samples from several patients. Distinct pathophysiological features of asthma can be

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients.

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Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

2015-10-30

Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. Copyright © 2015 Elsevier Inc. All rights reserved.

Investigation of Pokemon-regulated proteins in hepatocellular carcinoma using mass spectrometry-based multiplex quantitative proteomics.

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Bi, Xin; Jin, Yibao; Gao, Xiang; Liu, Feng; Gao, Dan; Jiang, Yuyang; Liu, Hongxia

2013-01-01

Pokemon is a transcription regulator involved in embryonic development, cellular differentiation and oncogenesis. It is aberrantly overexpressed in multiple human cancers including Hepatocellular carcinoma (HCC) and is considered as a promising biomarker for HCC. In this work, the isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics strategy was used to investigate the proteomic profile associated with Pokemon in human HCC cell line QGY7703 and human hepatocyte line HL7702. Samples were labeled with four-plex iTRAQ reagents followed by two-dimensional liquid chromatography coupled with tandem mass spectrometry analysis. A total of 24 differentially expressed proteins were selected as significant. Nine proteins were potentially up-regulated by Pokemon while 15 proteins were potentially down-regulated and many proteins were previously identified as potential biomarkers for HCC. Gene ontology (GO) term enrichment revealed that the listed proteins were mainly involved in DNA metabolism and biosynthesis process. The changes of glucose-6-phosphate 1-dehydrogenase (G6PD, up-regulated) and ribonucleoside-diphosphate reductase large sub-unit (RIM1, down-regulated) were validated by Western blotting analysis and denoted as Pokemon's function of oncogenesis. We also found that Pokemon potentially repressed the expression of highly clustered proteins (MCM3, MCM5, MCM6, MCM7) which played key roles in promoting DNA replication. Altogether, our results may help better understand the role of Pokemon in HCC and promote the clinical applications.

Comparative proteomic analysis reveals heart toxicity induced by chronic arsenic exposure in rats

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Huang, Qingyu; Xi, Guochen; Alamdar, Ambreen; Zhang, Jie; Shen, Heqing

2017-01-01

Arsenic is a widespread metalloid in the environment, which poses a broad spectrum of adverse effects on human health. However, a global view of arsenic-induced heart toxicity is still lacking, and the underlying molecular mechanisms remain unclear. By performing a comparative quantitative proteomic analysis, the present study aims to investigate the alterations of proteome profile in rat heart after long-term exposure to arsenic. As a result, we found that the abundance of 81 proteins were significantly altered by arsenic treatment (35 up-regulated and 46 down-regulated). Among these, 33 proteins were specifically associated with cardiovascular system development and function, including heart development, heart morphology, cardiac contraction and dilation, and other cardiovascular functions. It is further proposed that the aberrant regulation of 14 proteins induced by arsenic would disturb cardiac contraction and relaxation, impair heart morphogenesis and development, and induce thrombosis in rats, which is mediated by the Akt/p38 MAPK signaling pathway. Overall, these findings will augment our knowledge of the involved mechanisms and develop useful biomarkers for cardiotoxicity induced by environmental arsenic exposure. - Highlights: • Arsenic exposure has been associated with a number of adverse health effects. • The molecular mechanisms involved in arsenic-induced cardiotoxicity remain unclear. • Differential proteins were identified in arsenic-exposed rat heart by proteomics. • Arsenic induces heart toxicity through the Akt/p38 MAPK signaling pathway. - Label-free quantitative proteomic analysis of rat heart reveals putative mechanisms and biomarkers for arsenic-induced cardiotoxicity.

A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study

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Haußmann Ute

2011-06-01

Full Text Available Abstract Background Mass spectrometry-based proteomics has reached a stage where it is possible to comprehensively analyze the whole proteome of a cell in one experiment. Here, the employment of stable isotopes has become a standard technique to yield relative abundance values of proteins. In recent times, more and more experiments are conducted that depict not only a static image of the up- or down-regulated proteins at a distinct time point but instead compare developmental stages of an organism or varying experimental conditions. Results Although the scientific questions behind these experiments are of course manifold, there are, nevertheless, two questions that commonly arise: 1 which proteins are differentially regulated regarding the selected experimental conditions, and 2 are there groups of proteins that show similar abundance ratios, indicating that they have a similar turnover? We give advice on how these two questions can be answered and comprehensively compare a variety of commonly applied computational methods and their outcomes. Conclusions This work provides guidance through the jungle of computational methods to analyze mass spectrometry-based isotope-labeled datasets and recommends an effective and easy-to-use evaluation strategy. We demonstrate our approach with three recently published datasets on Bacillus subtilis 12 and Corynebacterium glutamicum 3. Special focus is placed on the application and validation of cluster analysis methods. All applied methods were implemented within the rich internet application QuPE 4. Results can be found at http://qupe.cebitec.uni-bielefeld.de.

Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

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Barkla, Bronwyn J; Vera-Estrella, Rosario; Raymond, Carolyn

2016-05-10

Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant. In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance. This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

iTRAQ-based quantitative proteomic analysis reveals proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum) in response to drought stress.

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Xie, He; Yang, Da-Hai; Yao, Heng; Bai, Ge; Zhang, Yi-Han; Xiao, Bing-Guang

2016-01-15

Drought is one of the most severe forms of abiotic stresses that threaten the survival of plants, including crops. In turn, plants dramatically change their physiology to increase drought tolerance, including reconfiguration of proteomes. Here, we studied drought-induced proteomic changes in leaves of cultivated tobacco (Nicotiana tabacum), a solanaceous plant, using the isobaric tags for relative and absolute quantitation (iTRAQ)-based protein labeling technology. Of identified 5570 proteins totally, drought treatment increased and decreased abundance of 260 and 206 proteins, respectively, compared with control condition. Most of these differentially regulated proteins are involved in photosynthesis, metabolism, and stress and defense. Although abscisic acid (ABA) levels greatly increased in drought-treated tobacco leaves, abundance of detected ABA biosynthetic enzymes showed no obvious changes. In contrast, heat shock proteins (HSPs), thioredoxins, ascorbate-, glutathione-, and hydrogen peroxide (H2O2)-related proteins were up- or down-regulated in drought-treated tobacco leaves, suggesting that chaperones and redox signaling are important for tobacco tolerance to drought, and it is likely that redox-induced posttranslational modifications play an important role in modulating protein activity. This study not only provides a comprehensive dataset on overall protein changes in drought-treated tobacco leaves, but also shed light on the mechanism by which solanaceous plants adapt to drought stress. Copyright © 2015 Yunnan Academy of Tobacco Agricultural Sciences. Published by Elsevier Inc. All rights reserved.

Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring.

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Song, Jun; Du, Lina; Li, Li; Kalt, Wilhelmina; Palmer, Leslie Campbell; Fillmore, Sherry; Zhang, Ying; Zhang, ZhaoQi; Li, XiHong

2015-06-03

To better understand the regulation of flavonoid and anthocyanin biosynthesis, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars at three ripening stages. This quantitative proteomic workflow was improved through an OFFGEL electrophoresis to fractionate peptides from total protein digests. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were investigated. The normalized protein abundance, which was measured using isotopically-labeled standards, was significantly changed concurrently with increased anthocyanin content and advanced fruit maturity. The protein abundance of phenylalanine ammonia-lyase; anthocyanidin synthase, chalcone isomerase; flavanone 3-hydroxylase; dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2, was all significantly increased in fruit of more advanced ripeness. An interaction between cultivar and maturity was also shown with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin content suggested that a metabolic control point may exist for anthocyanin biosynthesis. This research provides insights into the process of anthocyanin formation in strawberry fruit at the level of protein concentration and reveals possible candidates in the regulation of anthocyanin formation during fruit ripening. To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides

Protein profiling in hepatocellular carcinoma by label-free quantitative proteomics in two west African populations.

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Haddy K S Fye

Full Text Available Hepatocellular Carcinoma is the third most common cause of cancer related death worldwide, often diagnosed by measuring serum AFP; a poor performance stand-alone biomarker. With the aim of improving on this, our study focuses on plasma proteins identified by Mass Spectrometry in order to investigate and validate differences seen in the respective proteomes of controls and subjects with LC and HCC.Mass Spectrometry analysis using liquid chromatography electro spray ionization quadrupole time-of-flight was conducted on 339 subjects using a pooled expression profiling approach. ELISA assays were performed on four significantly differentially expressed proteins to validate their expression profiles in subjects from the Gambia and a pilot group from Nigeria. Results from this were collated for statistical multiplexing using logistic regression analysis.Twenty-six proteins were identified as differentially expressed between the three subject groups. Direct measurements of four; hemopexin, alpha-1-antitrypsin, apolipoprotein A1 and complement component 3 confirmed their change in abundance in LC and HCC versus control patients. These trends were independently replicated in the pilot validation subjects from Nigeria. The statistical multiplexing of these proteins demonstrated performance comparable to or greater than ALT in identifying liver cirrhosis or carcinogenesis. This exercise also proposed preliminary cut offs with achievable sensitivity, specificity and AUC statistics greater than reported AFP averages.The validated changes of expression in these proteins have the potential for development into high-performance tests usable in the diagnosis and or monitoring of HCC and LC patients. The identification of sustained expression trends strengthens the suggestion of these four proteins as worthy candidates for further investigation in the context of liver disease. The statistical combinations also provide a novel inroad of analyses able to propose

Differential alkylation-based redox proteomics--Lessons learnt.

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Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina

2015-12-01

Cysteine is one of the most reactive amino acids. This is due to the electronegativity of sulphur atom in the side chain of thiolate group. It results in cysteine being present in several distinct redox forms inside the cell. Amongst these, reversible oxidations, S-nitrosylation and S-sulfenylation are crucial mediators of intracellular redox signalling, with known associations to health and disease. Study of their functionalities has intensified thanks to the development of various analytical strategies, with particular contribution from differential alkylation-based proteomics methods. Presented here is a critical evaluation of differential alkylation-based strategies for the analysis of S-nitrosylation and S-sulfenylation. The aim is to assess the current status and to provide insights for future directions in the dynamically evolving field of redox proteomics. To achieve that we collected 35 original research articles published since 2010 and analysed them considering the following parameters, (i) resolution of modification site, (ii) quantitative information, including correction of modification levels by protein abundance changes and determination of modification site occupancy, (iii) throughput, including the amount of starting material required for analysis. The results of this meta-analysis are the core of this review, complemented by issues related to biological models and sample preparation in redox proteomics, including conditions for free thiol blocking and labelling of target cysteine oxoforms. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Redefining the Breast Cancer Exosome Proteome by Tandem Mass Tag Quantitative Proteomics and Multivariate Cluster Analysis.

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Clark, David J; Fondrie, William E; Liao, Zhongping; Hanson, Phyllis I; Fulton, Amy; Mao, Li; Yang, Austin J

2015-10-20

Exosomes are microvesicles of endocytic origin constitutively released by multiple cell types into the extracellular environment. With evidence that exosomes can be detected in the blood of patients with various malignancies, the development of a platform that uses exosomes as a diagnostic tool has been proposed. However, it has been difficult to truly define the exosome proteome due to the challenge of discerning contaminant proteins that may be identified via mass spectrometry using various exosome enrichment strategies. To better define the exosome proteome in breast cancer, we incorporated a combination of Tandem-Mass-Tag (TMT) quantitative proteomics approach and Support Vector Machine (SVM) cluster analysis of three conditioned media derived fractions corresponding to a 10 000g cellular debris pellet, a 100 000g crude exosome pellet, and an Optiprep enriched exosome pellet. The quantitative analysis identified 2 179 proteins in all three fractions, with known exosomal cargo proteins displaying at least a 2-fold enrichment in the exosome fraction based on the TMT protein ratios. Employing SVM cluster analysis allowed for the classification 251 proteins as "true" exosomal cargo proteins. This study provides a robust and vigorous framework for the future development of using exosomes as a potential multiprotein marker phenotyping tool that could be useful in breast cancer diagnosis and monitoring disease progression.

Label-Free Quantitation of Ribosomal Proteins from Bacillus subtilis for Antibiotic Research.

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Schäkermann, Sina; Prochnow, Pascal; Bandow, Julia E

2017-01-01

Current research is focusing on ribosome heterogeneity as a response to changing environmental conditions and stresses, such as antibiotic stress. Altered stoichiometry and composition of ribosomal proteins as well as association of additional protein factors are mechanisms for shaping the protein expression profile or hibernating ribosomes. Here, we present a method for the isolation of ribosomes to analyze antibiotic-induced changes in the composition of ribosomes in Bacillus subtilis or other bacteria. Ribosomes and associated proteins are isolated by ultracentrifugation and proteins are identified and quantified using label-free mass spectrometry.

Halobacterium salinarum NRC-1 PeptideAtlas: toward strategies for targeted proteomics and improved proteome coverage.

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Van, Phu T; Schmid, Amy K; King, Nichole L; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T; Goo, Young Ah; Deutsch, Eric W; Reiss, David J; Mallick, Parag; Baliga, Nitin S

2008-09-01

The relatively small numbers of proteins and fewer possible post-translational modifications in microbes provide a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a PeptideAtlas (PA) covering 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636 000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has highlighted plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore, we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics.

Quantitative proteomic analysis for novel biomarkers of buccal squamous cell carcinoma arising in background of oral submucous fibrosis

International Nuclear Information System (INIS)

Liu, Wen; Zeng, Lijuan; Li, Ning; Wang, Fei; Jiang, Canhua; Guo, Feng; Chen, Xinqun; Su, Tong; Xu, Chunjiao; Zhang, Shanshan; Fang, Changyun

2016-01-01

In South and Southeast Asian, the majority of buccal squamous cell carcinoma (BSCC) can arise from oral submucous fibrosis (OSF). BSCCs develop in OSF that are often not completely resected, causing local relapse. The aim of our study was to find candidate protein biomarkers to detect OSF and predict prognosis in BSCCs by quantitative proteomics approaches. We compared normal oral mucosa (NBM) and paired biopsies of BSCC and OSF by quantitative proteomics using isobaric tags for relative and absolute quantification (iTRAQ) to discover proteins with differential expression. Gene Ontology and KEGG networks were analyzed. The prognostic value of biomarkers was evaluated in 94 BSCCs accompanied with OSF. Significant associations were assessed by Kaplan-Meier survival and Cox-proportional hazards analysis. In total 30 proteins were identified with significantly different expression (false discovery rate < 0.05) among three tissues. Two consistently upregulated proteins, ANXA4 and FLNA, were validated. The disease-free survival was negatively associated with the expression of ANXA4 (hazard ratio, 3.4; P = 0.000), FLNA (hazard ratio, 2.1; P = 0.000) and their combination (hazard ratio, 8.8; P = 0.002) in BSCCs. The present study indicates that iTRAQ quantitative proteomics analysis for tissues of BSCC and OSF is a reliable strategy. A significantly up-regulated ANXA4 and FLNA could be not only candidate biomarkers for BSCC prognosis but also potential targets for its therapy. The online version of this article (doi:10.1186/s12885-016-2650-1) contains supplementary material, which is available to authorized users

Comparative proteomic analysis of human malignant ascitic fluids for the development of gastric cancer biomarkers.

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Jin, Jonghwa; Son, Minsoo; Kim, Hyeyoon; Kim, Hyeyeon; Kong, Seong-Ho; Kim, Hark Kyun; Kim, Youngsoo; Han, Dohyun

2018-04-11

Malignant ascites is a sign of peritoneal seeding, which is one of the most frequent forms of incurable distant metastasis. Because the development of malignant ascites is associated with an extremely poor prognosis, determining whether it resulted from peritoneal seeding has critical clinical implications in diagnosis, choice of treatment, and active surveillance. At present, the molecular characterizations of malignant ascites are especially limited in case of gastric cancer. We aimed to identify malignant ascites-specific proteins that may contribute to the development of alternative methods for diagnosis and therapeutic monitoring and also increase our understanding of the pathophysiology of peritoneal seeding. First, comprehensive proteomic strategies were employed to construct an in-depth proteome of ascitic fluids. Label-free quantitative proteomic analysis was subsequently performed to identify candidates that can differentiate between malignant ascitic fluilds of gastric cancer patients from benign ascitic fluids. Finally, two candidate proteins were verified by ELISA in 84 samples with gastric cancer or liver cirrhosis. Comprehensive proteome profiling resulted in the identification of 5347 ascites proteins. Using label-free quantification, we identified 299 proteins that were differentially expressed in ascitic fluids between liver cirrhosis and stage IV gastric cancer patients. In addition, we identified 645 proteins that were significantly expressed in ascitic fluids between liver cirrhosis and gastric cancer patients with peritoneal seeding. Finally, Gastriscin and Periostin that can distinguish malignant ascites from benign ascites were verified by ELISA. This study identified and verified protein markers that can distinguish malignant ascites with or without peritoneal seeding from benign ascites. Consequently, our results could be a significant resource for gastric cancer research and biomarker discovery in the diagnosis of malignant ascites

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

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Mu, Jun [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Yang, Yongtao [Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China); Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Xie, Peng, E-mail: xiepeng@cqmu.edu.cn [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing (China); Institute of Neuroscience and the Collaborative Innovation Center for Brain Science, Chongqing Medical University, Chongqing (China); Chongqing Key Laboratory of Neurobiology, Chongqing (China); Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing (China)

2015-10-30

Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

Elevated host lipid metabolism revealed by iTRAQ-based quantitative proteomic analysis of cerebrospinal fluid of tuberculous meningitis patients

International Nuclear Information System (INIS)

Mu, Jun; Yang, Yongtao; Chen, Jin; Cheng, Ke; Li, Qi; Wei, Yongdong; Zhu, Dan; Shao, Weihua; Zheng, Peng; Xie, Peng

2015-01-01

Purpose: Tuberculous meningitis (TBM) remains to be one of the most deadly infectious diseases. The pathogen interacts with the host immune system, the process of which is largely unknown. Various cellular processes of Mycobacterium tuberculosis (MTB) centers around lipid metabolism. To determine the lipid metabolism related proteins, a quantitative proteomic study was performed here to identify differential proteins in the cerebrospinal fluid (CSF) obtained from TBM patients (n = 12) and healthy controls (n = 12). Methods: CSF samples were desalted, concentrated, labelled with isobaric tags for relative and absolute quantitation (iTRAQ™), and analyzed by multi-dimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene ontology and proteomic phenotyping analysis of the differential proteins were conducted using Database for Annotation, Visualization, and Integrated Discovery (DAVID) Bioinformatics Resources. ApoE and ApoB were selected for validation by ELISA. Results: Proteomic phenotyping of the 4 differential proteins was invloved in the lipid metabolism. ELISA showed significantly increased ApoB levels in TBM subjects compared to healthy controls. Area under the receiver operating characteristic curve analysis demonstrated ApoB levels could distinguish TBM subjects from healthy controls and viral meningitis subjects with 89.3% sensitivity and 92% specificity. Conclusions: CSF lipid metabolism disregulation, especially elevated expression of ApoB, gives insights into the pathogenesis of TBM. Further evaluation of these findings in larger studies including anti-tuberculosis medicated and unmedicated patient cohorts with other center nervous system infectious diseases is required for successful clinical translation. - Highlights: • The first proteomic study on the cerebrospinal fluid of tuberculous meningitis patients using iTRAQ. • Identify 4 differential proteins invloved in the lipid metabolism. • Elevated expression of ApoB gives

Proteomics of Eosinophil Activation

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Deane F. Mosher

2017-09-01

Full Text Available We recently identified and quantified >7,000 proteins in non-activated human peripheral blood eosinophils using liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS and described phosphoproteomic changes that accompany acute activation of eosinophils by interleukin-5 (IL5 (1. These data comprise a treasure trove of information about eosinophils. We illustrate the power of label-free LC–MS/MS quantification by considering four examples: complexity of eosinophil STATs, contribution of immunoproteasome subunits to eosinophil proteasomes, complement of integrin subunits, and contribution of platelet proteins originating from platelet–eosinophil complexes to the overall proteome. We describe how isobaric labeling enables robust sample-to-sample comparisons and relate the 220 phosphosites that changed significantly upon treatment with IL5 to previous studies of eosinophil activation. Finally, we review previous attempts to leverage the power of mass spectrometry to discern differences between eosinophils of healthy subjects and those with eosinophil-associated conditions and point out features of label-free quantification and isobaric labeling that are important in planning future mass spectrometric studies.

iTRAQ Quantitative Proteomic Analysis of Vitreous from Patients with Retinal Detachment

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Fátima Milhano Santos

2018-04-01

Full Text Available Rhegmatogenous retinal detachment (RRD is a potentially blinding condition characterized by a physical separation between neurosensory retina and retinal pigment epithelium. Quantitative proteomics can help to understand the changes that occur at the cellular level during RRD, providing additional information about the molecular mechanisms underlying its pathogenesis. In the present study, iTRAQ labeling was combined with two-dimensional LC-ESI-MS/MS to find expression changes in the proteome of vitreous from patients with RRD when compared to control samples. A total of 150 proteins were found differentially expressed in the vitreous of patients with RRD, including 96 overexpressed and 54 underexpressed. Several overexpressed proteins, several such as glycolytic enzymes (fructose-bisphosphate aldolase A, gamma-enolase, and phosphoglycerate kinase 1, glucose transporters (GLUT-1, growth factors (metalloproteinase inhibitor 1, and serine protease inhibitors (plasminogen activator inhibitor 1 are regulated by HIF-1, which suggests that HIF-1 signaling pathway can be triggered in response to RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Also, the accumulation of photoreceptor proteins, including phosducin, rhodopsin, and s-arrestin, and vimentin in vitreous may indicate that photoreceptor degeneration occurs in RRD. Nevertheless, the differentially expressed proteins found in this study suggest that different mechanisms are activated after RRD to promote the survival of retinal cells through complex cellular responses.

Data Generated by Quantitative Liquid Chromatography-Mass Spectrometry Proteomics Are Only the Start and Not the Endpoint: Optimization of Quantitative Concatemer-Based Measurement of Hepatic Uridine-5'-Diphosphate-Glucuronosyltransferase Enzymes with Reference to Catalytic Activity.

Science.gov (United States)

Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Al-Majdoub, Zubida M; Goosen, Theunis C; Rostami-Hodjegan, Amin; Barber, Jill

2018-06-01

Quantitative proteomic methods require optimization at several stages, including sample preparation, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and data analysis, with the final analysis stage being less widely appreciated by end-users. Previously reported measurement of eight uridine-5'-diphospho-glucuronosyltransferases (UGT) generated by two laboratories [using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT)] reflected significant disparity between proteomic methods. Initial analysis of QconCAT data showed lack of correlation with catalytic activity for several UGTs (1A4, 1A6, 1A9, 2B15) and moderate correlations for UGTs 1A1, 1A3, and 2B7 ( R s = 0.40-0.79, P data analysis, starting from unprocessed LC-MS/MS data, was undertaken, with the aim of improving accuracy, defined by correlation against activity. Three main criteria were found to be important: choice of monitored peptides and fragments, correction for isotope-label incorporation, and abundance normalization using fractional protein mass. Upon optimization, abundance-activity correlations improved significantly for six UGTs ( R s = 0.53-0.87, P data analysis strategy and indicates, using examples, the significance of systematic data processing following acquisition. The proposed strategy offers significant improvement on existing guidelines applicable to clinically relevant proteins quantified using QconCAT. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

Quantitative proteomic analysis for high-throughput screening of differential glycoproteins in hepatocellular carcinoma serum

International Nuclear Information System (INIS)

Gao, Hua-Jun; Chen, Ya-Jing; Zuo, Duo; Xiao, Ming-Ming; Li, Ying; Guo, Hua; Zhang, Ning; Chen, Rui-Bing

2015-01-01

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths. Novel serum biomarkers are required to increase the sensitivity and specificity of serum screening for early HCC diagnosis. This study employed a quantitative proteomic strategy to analyze the differential expression of serum glycoproteins between HCC and normal control serum samples. Lectin affinity chromatography (LAC) was used to enrich glycoproteins from the serum samples. Quantitative mass spectrometric analysis combined with stable isotope dimethyl labeling and 2D liquid chromatography (LC) separations were performed to examine the differential levels of the detected proteins between HCC and control serum samples. Western blot was used to analyze the differential expression levels of the three serum proteins. A total of 2,280 protein groups were identified in the serum samples from HCC patients by using the 2D LC-MS/MS method. Up to 36 proteins were up-regulated in the HCC serum, whereas 19 proteins were down-regulated. Three differential glycoproteins, namely, fibrinogen gamma chain (FGG), FOS-like antigen 2 (FOSL2), and α-1,6-mannosylglycoprotein 6-β-N-acetylglucosaminyltransferase B (MGAT5B) were validated by Western blot. All these three proteins were up-regulated in the HCC serum samples. A quantitative glycoproteomic method was established and proven useful to determine potential novel biomarkers for HCC

Proteome identification of the silkworm middle silk gland

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Jian-ying Li

2016-03-01

Full Text Available To investigate the functional differentiation among the anterior (A, middle (M, and posterior (P regions of silkworm middle silk gland (MSG, their proteomes were characterized by shotgun LC–MS/MS analysis with a LTQ-Orbitrap mass spectrometer. To get better proteome identification and quantification, triplicate replicates of mass spectrometry analysis were performed for each sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (Vizcaíno et al., 2014 [1] via the PRIDE partner repository (Vizcaino, 2013 [2] with the dataset identifier http://www.ebi.ac.uk/pride/archive/projects/PXD003371. The peptide identifications that were further processed by PeptideProphet program in Trans-Proteomic Pipeline (TPP after database search with Mascot software were also available in .XML format files. Data presented here are related to a research article published in Journal of Proteomics by Li et al. (2015 [3]. Keywords: Bombyx mori, Middle silk gland, Silk protein synthesis, Shotgun proteomics, Label-free

A miniaturized optoelectronic system for rapid quantitative label-free detection of harmful species in food

Science.gov (United States)

Raptis, Ioannis; Misiakos, Konstantinos; Makarona, Eleni; Salapatas, Alexandros; Petrou, Panagiota; Kakabakos, Sotirios; Botsialas, Athanasios; Jobst, Gerhard; Haasnoot, Willem; Fernandez-Alba, Amadeo; Lees, Michelle; Valamontes, Evangelos

2016-03-01

Optical biosensors have emerged in the past decade as the most promising candidates for portable, highly-sensitive bioanalytical systems that can be employed for in-situ measurements. In this work, a miniaturized optoelectronic system for rapid, quantitative, label-free detection of harmful species in food is presented. The proposed system has four distinctive features that can render to a powerful tool for the next generation of Point-of-Need applications, namely it accommodates the light sources and ten interferometric biosensors on a single silicon chip of a less-than-40mm2 footprint, each sensor can be individually functionalized for a specific target analyte, the encapsulation can be performed at the wafer-scale, and finally it exploits a new operation principle, Broad-band Mach-Zehnder Interferometry to ameliorate its analytical capabilities. Multi-analyte evaluation schemes for the simultaneous detection of harmful contaminants, such as mycotoxins, allergens and pesticides, proved that the proposed system is capable of detecting within short time these substances at concentrations below the limits imposed by regulatory authorities, rendering it to a novel tool for the near-future food safety applications.

High-throughput, label-free, single-cell, microalgal lipid screening by machine-learning-equipped optofluidic time-stretch quantitative phase microscopy.

Science.gov (United States)

Guo, Baoshan; Lei, Cheng; Kobayashi, Hirofumi; Ito, Takuro; Yalikun, Yaxiaer; Jiang, Yiyue; Tanaka, Yo; Ozeki, Yasuyuki; Goda, Keisuke

2017-05-01

The development of reliable, sustainable, and economical sources of alternative fuels to petroleum is required to tackle the global energy crisis. One such alternative is microalgal biofuel, which is expected to play a key role in reducing the detrimental effects of global warming as microalgae absorb atmospheric CO 2 via photosynthesis. Unfortunately, conventional analytical methods only provide population-averaged lipid amounts and fail to characterize a diverse population of microalgal cells with single-cell resolution in a non-invasive and interference-free manner. Here high-throughput label-free single-cell screening of lipid-producing microalgal cells with optofluidic time-stretch quantitative phase microscopy was demonstrated. In particular, Euglena gracilis, an attractive microalgal species that produces wax esters (suitable for biodiesel and aviation fuel after refinement), within lipid droplets was investigated. The optofluidic time-stretch quantitative phase microscope is based on an integration of a hydrodynamic-focusing microfluidic chip, an optical time-stretch quantitative phase microscope, and a digital image processor equipped with machine learning. As a result, it provides both the opacity and phase maps of every single cell at a high throughput of 10,000 cells/s, enabling accurate cell classification without the need for fluorescent staining. Specifically, the dataset was used to characterize heterogeneous populations of E. gracilis cells under two different culture conditions (nitrogen-sufficient and nitrogen-deficient) and achieve the cell classification with an error rate of only 2.15%. The method holds promise as an effective analytical tool for microalgae-based biofuel production. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Quantitative proteomic analysis of whey proteins in the colostrum and mature milk of yak (Bos grunniens).

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Yang, Yongxin; Zhao, Xiaowei; Yu, Shumin; Cao, Suizhong

2015-02-01

Yak (Bos grunniens) is an important natural resource in mountainous regions. To date, few studies have addressed the differences in the protein profiles of yak colostrum and milk. We used quantitative proteomics to compare the protein profiles of whey from yak colostrum and milk. Milk samples were collected from 21 yaks after calving (1 and 28 d). Whey protein profiles were generated through isobaric tag for relative and absolute quantification (iTRAQ)-labelled proteomics. We identified 183 proteins in milk whey; of these, the expression levels of 86 proteins differed significantly between the whey from colostrum and milk. Haemoglobin expression showed the greatest change; its levels were significantly higher in the whey from colostrum than in mature milk whey. Functional analysis revealed that many of the differentially expressed proteins were associated with biological regulation and response to stimuli. Further, eight differentially expressed proteins involved in the complement and coagulation cascade pathway were enriched in milk whey. These findings add to the general understanding of the protein composition of yak milk, suggest potential functions of the differentially expressed proteins, and provide novel information on the role of colostral components in calf survival. © 2014 Society of Chemical Industry.

6. Label-free selective plane illumination microscopy of tissue samples

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Muteb Alharbi

2017-10-01

Conclusion: Overall this method meets the demands of the current needs for 3D imaging tissue samples in a label-free manner. Label-free Selective Plane Microscopy directly provides excellent information about the structure of the tissue samples. This work has highlighted the superiority of Label-free Selective Plane Microscopy to current approaches to label-free 3D imaging of tissue.

Combinatorial hexapeptide ligand libraries (ProteoMiner): an innovative fractionation tool for differential quantitative clinical proteomics.

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Hartwig, Sonja; Czibere, Akos; Kotzka, Jorg; Passlack, Waltraud; Haas, Rainer; Eckel, Jürgen; Lehr, Stefan

2009-07-01

Blood serum samples are the major source for clinical proteomics approaches, which aim to identify diagnostically relevant or treatment-response related proteins. But, the presence of very high-abundance proteins and the enormous dynamic range of protein distribution hinders whole serum analysis. An innovative tool to overcome these limitations, utilizes combinatorial hexapeptide ligand libraries (ProteoMiner). Here, we demonstrate that ProteoMiner can be used for comparative and quantitative analysis of complex proteomes. We spiked serum samples with increasing amounts (3 microg to 300 microg) of whole E. coli lysate, processed it with ProteoMiner and performed quantitative analyses of 2D-gels. We found, that the concentration of the spiked bacteria proteome, reflected by the maintained proportional spot intensities, was not altered by ProteoMiner treatment. Therefore, we conclude that the ProteoMiner technology can be used for quantitative analysis of low abundant proteins in complex biological samples.

Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive.

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Lebesgue, Nicolas; da Costa, Gonçalo; Ribeiro, Raquel Mesquita; Ribeiro-Silva, Cristina; Martins, Gabriel G; Matranga, Valeria; Scholten, Arjen; Cordeiro, Carlos; Heck, Albert J R; Santos, Romana

2016-06-01

Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc) versus the non-adhesive part (the stem), and also to profile the proteome of the secreted adhesive (glue). This data article contains complementary figures and results related to the research article "Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach" (Lebesgue et al., 2016) [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold), likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives.

Proteomic dataset of the sea urchin Paracentrotus lividus adhesive organs and secreted adhesive

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Nicolas Lebesgue

2016-06-01

Full Text Available Sea urchins have specialized adhesive organs called tube feet, which mediate strong but reversible adhesion. Tube feet are composed by a disc, producing adhesive and de-adhesive secretions for substratum attachment, and a stem for movement. After detachment the secreted adhesive remains bound to the substratum as a footprint. Recently, a label-free quantitative proteomic approach coupled with the latest mass-spectrometry technology was used to analyze the differential proteome of Paracentrotus lividus adhesive organ, comparing protein expression levels in the tube feet adhesive part (the disc versus the non-adhesive part (the stem, and also to profile the proteome of the secreted adhesive (glue. This data article contains complementary figures and results related to the research article “Deciphering the molecular mechanisms underlying sea urchin reversible adhesion: a quantitative proteomics approach” (Lebesgue et al., 2016 [1]. Here we provide a dataset of 1384 non-redundant proteins, their fragmented peptides and expression levels, resultant from the analysis of the tube feet differential proteome. Of these, 163 highly over-expressed tube feet disc proteins (>3-fold, likely representing the most relevant proteins for sea urchin reversible adhesion, were further annotated in order to determine the potential functions. In addition, we provide a dataset of 611 non-redundant proteins identified in the secreted adhesive proteome, as well as their functional annotation and grouping in 5 major protein groups related with adhesive exocytosis, and microbial protection. This list was further analyzed to identify the most abundant protein groups and pinpoint putative adhesive proteins, such as Nectin, the most abundant adhesive protein in sea urchin glue. The obtained data uncover the key proteins involved in sea urchins reversible adhesion, representing a step forward to the development of new wet-effective bio-inspired adhesives.

Impact of phenolic substrate and growth temperature on the arthrobacter chlorophenolicus proteome

Energy Technology Data Exchange (ETDEWEB)

Unell, Maria; Abraham, Paul E.; Shah, Manesh; Zhang, Bing; Ruckert, Christian; VerBerkmoes, Nathan C.; Jansson, Janet K.

2009-02-15

We compared the Arthrobacter chlorophenolicus proteome during growth on 4-chlorophenol, 4-nitrophenol or phenol at 5 C and 28 C; both for the wild type and a mutant strain with mass spectrometry based proteomics. A label free workflow employing spectral counting identified 3749 proteins across all growth conditions, representing over 70% of the predicted genome and 739 of these proteins form the core proteome. Statistically significant differences were found in the proteomes of cells grown under different conditions including differentiation of hundreds of unknown proteins. The 4-chlorophenol-degradation pathway was confirmed, but not that for phenol.

Quantitative proteomic characterization of redox-dependent post-translational modifications on protein cysteines

Energy Technology Data Exchange (ETDEWEB)

Duan, Jicheng; Gaffrey, Matthew J.; Qian, Wei-Jun

2017-01-01

Protein cysteine thiols play a crucial role in redox signaling, regulation of enzymatic activity and protein function, and maintaining redox homeostasis in living systems. The unique chemical reactivity of thiol groups makes cysteine susceptible to oxidative modifications by reactive oxygen and nitrogen species to form a broad array of reversible and irreversible protein post-translational modifications (PTMs). The reversible modifications in particular are one of the major components of redox signaling and are involved in regulation of various cellular processes under physiological and pathological conditions. The biological significance of these redox PTMs in health and diseases has been increasingly recognized. Herein, we review the recent advances of quantitative proteomic approaches for investigating redox PTMs in complex biological systems, including the general considerations of sample processing, various chemical or affinity enrichment strategies, and quantitative approaches. We also highlight a number of redox proteomic approaches that enable effective profiling of redox PTMs for addressing specific biological questions. Although some technological limitations remain, redox proteomics is paving the way towards a better understanding of redox signaling and regulation in human health and diseases.

Proteomic profiling of Mycobacterium tuberculosis identifies nutrient-starvation-responsive toxin-antitoxin systems

DEFF Research Database (Denmark)

Albrethsen, Jakob; Agner, Jeppe; Piersma, Sander R

2013-01-01

In order to successfully enter the latent stage, Mycobacterium tuberculosis must adapt to conditions such as nutrient limitation and hypoxia. In vitro models that mimic latent infection are valuable tools for describing the changes in metabolism that occur when the bacterium exists in a non......-growing form. We used two complementary proteomic approaches, label-free LC-MS/MS analysis and two-dimensional difference gel electrophoresis, to determine the proteome profile of extracellular proteins from M. tuberculosis cultured under nutrient starvation. Through the label-free LC-MS/MS analysis......, significant differences in the overall metabolism during nutrient starvation were detected. Notably, members of the toxin-antitoxin systems were present in larger quantities in nutrient-starved cultures, supporting a role for these global modules as M. tuberculosis switches its metabolism into dormancy...

Differential membrane proteomics using 18O-labeling to identify biomarkers for cholangiocarcinoma

DEFF Research Database (Denmark)

Kristiansen, Troels Zakarias; Harsha, H C; Grønborg, Mads

2008-01-01

Quantitative proteomic methodologies allow profiling of hundreds to thousands of proteins in a high-throughput fashion. This approach is increasingly applied to cancer biomarker discovery to identify proteins that are differentially regulated in cancers. Fractionation of protein samples based...

Proteomic profile response of Paracoccidioides lutzii to the antifungal argentilactone

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Renata Silva Do Prado

2015-06-01

Full Text Available The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM, a mycosis of high incidence in Brazil. The toxicity of drug treatment and the emergence of resistant organisms have led to research for new candidates for drugs. In this study, we demonstrate that the natural product argentilactone was not cytotoxic or genotoxic to MRC5 cells at the IC50 concentration to the fungus. We also verified the proteomic profile of Paracoccidioides lutzii after incubation with argentilactone using a label free quantitative proteome nanoUPLC-MSE. The results of this study indicated that the fungus has a global metabolic adaptation in the presence of argentilactone. Enzymes of important pathways, such as glycolysis, the Krebs cycle and the glyoxylate cycle, were repressed, which drove the metabolism to the methylcytrate cycle and beta-oxidation. Proteins involved in cell rescue, defense and stress response were induced. In this study, alternative metabolic pathways adopted by the fungi were elucidated, helping to elucidate the course of action of the compound studied.

Shotgun proteomics of plant plasma membrane and microdomain proteins using nano-LC-MS/MS.

Science.gov (United States)

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

2014-01-01

Shotgun proteomics allows the comprehensive analysis of proteins extracted from plant cells, subcellular organelles, and membranes. Previously, two-dimensional gel electrophoresis-based proteomics was used for mass spectrometric analysis of plasma membrane proteins. In order to get comprehensive proteome profiles of the plasma membrane including highly hydrophobic proteins with a number of transmembrane domains, a mass spectrometry-based shotgun proteomics method using nano-LC-MS/MS for proteins from the plasma membrane proteins and plasma membrane microdomain fraction is described. The results obtained are easily applicable to label-free protein semiquantification.

Differential alkylation-based redox proteomics – Lessons learnt

Science.gov (United States)

Wojdyla, Katarzyna; Rogowska-Wrzesinska, Adelina

2015-01-01

Cysteine is one of the most reactive amino acids. This is due to the electronegativity of sulphur atom in the side chain of thiolate group. It results in cysteine being present in several distinct redox forms inside the cell. Amongst these, reversible oxidations, S-nitrosylation and S-sulfenylation are crucial mediators of intracellular redox signalling, with known associations to health and disease. Study of their functionalities has intensified thanks to the development of various analytical strategies, with particular contribution from differential alkylation-based proteomics methods. Presented here is a critical evaluation of differential alkylation-based strategies for the analysis of S-nitrosylation and S-sulfenylation. The aim is to assess the current status and to provide insights for future directions in the dynamically evolving field of redox proteomics. To achieve that we collected 35 original research articles published since 2010 and analysed them considering the following parameters, (i) resolution of modification site, (ii) quantitative information, including correction of modification levels by protein abundance changes and determination of modification site occupancy, (iii) throughput, including the amount of starting material required for analysis. The results of this meta-analysis are the core of this review, complemented by issues related to biological models and sample preparation in redox proteomics, including conditions for free thiol blocking and labelling of target cysteine oxoforms. PMID:26282677

Quantitative Proteomic Analysis of Wheat Seeds during Artificial Ageing and Priming Using the Isobaric Tandem Mass Tag Labeling.

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Yangyong Lv

Full Text Available Wheat (Triticum aestivum L. is an important crop worldwide. The physiological deterioration of seeds during storage and seed priming is closely associated with germination, and thus contributes to plant growth and subsequent grain yields. In this study, wheat seeds during different stages of artificial ageing (45°C; 50% relative humidity; 98%, 50%, 20%, and 1% Germination rates and priming (hydro-priming treatment were subjected to proteomics analysis through a proteomic approach based on the isobaric tandem mass tag labeling. A total of 162 differentially expressed proteins (DEPs mainly involved in metabolism, energy supply, and defense/stress responses, were identified during artificial ageing and thus validated previous physiological and biochemical studies. These DEPs indicated that the inability to protect against ageing leads to the incremental decomposition of the stored substance, impairment of metabolism and energy supply, and ultimately resulted in seed deterioration. Kyoto Encyclopedia of Genes and Genomes (KEGG analysis revealed that the up-regulated proteins involved in seed ageing were mainly enriched in ribosome, whereas the down-regulated proteins were mainly accumulated in energy supply (starch and sucrose metabolism and stress defense (ascorbate and aldarate metabolism. Proteins, including hemoglobin 1, oleosin, agglutinin, and non-specific lipid-transfer proteins, were first identified in aged seeds and might be regarded as new markers of seed deterioration. Of the identified proteins, 531 DEPs were recognized during seed priming compared with unprimed seeds. In contrast to the up-regulated DEPs in seed ageing, several up-regulated DEPs in priming were involved in energy supply (tricarboxylic acid cycle, glycolysis, and fatty acid oxidation, anabolism (amino acids, and fatty acid synthesis, and cell growth/division. KEGG and protein-protein interaction analysis indicated that the up-regulated proteins in seed priming were

Progress of new label-free techniques for biosensors: a review.

Science.gov (United States)

Sang, Shengbo; Wang, Yajun; Feng, Qiliang; Wei, Ye; Ji, Jianlong; Zhang, Wendong

2016-01-01

The detection techniques used in biosensors can be broadly classified into label-based and label-free. Label-based detection relies on the specific properties of labels for detecting a particular target. In contrast, label-free detection is suitable for the target molecules that are not labeled or the screening of analytes which are not easy to tag. Also, more types of label-free biosensors have emerged with developments in biotechnology. The latest developed techniques in label-free biosensors, such as field-effect transistors-based biosensors including carbon nanotube field-effect transistor biosensors, graphene field-effect transistor biosensors and silicon nanowire field-effect transistor biosensors, magnetoelastic biosensors, optical-based biosensors, surface stress-based biosensors and other type of biosensors based on the nanotechnology are discussed. The sensing principles, configurations, sensing performance, applications, advantages and restriction of different label-free based biosensors are considered and discussed in this review. Most concepts included in this survey could certainly be applied to the development of this kind of biosensor in the future.

Label-free characterization of ultra violet-radiation-induced changes in skin fibroblasts with Raman spectroscopy and quantitative phase microscopy.

Science.gov (United States)

Singh, S P; Kang, Sungsam; Kang, Jeon Woong; So, Peter T C; Dasari, Ramanchandra Rao; Yaqoob, Zahid; Barman, Ishan

2017-09-07

Minimizing morbidities and mortalities associated with skin cancers requires sustained research with the goal of obtaining fresh insights into disease onset and progression under specific stimuli, particularly the influence of ultraviolet rays. In the present study, label-free profiling of skin fibroblasts exposed to time-bound ultra-violet radiation has been performed using quantitative phase imaging and Raman spectroscopy. Statistically significant differences in quantifiable biophysical parameters, such as matter density and cell dry mass, were observed with phase imaging. Accurate estimation of changes in the biochemical constituents, notably nucleic acids and proteins, was demonstrated through a combination of Raman spectroscopy and multivariate analysis of spectral patterns. Overall, the findings of this study demonstrate the promise of these non-perturbative optical modalities in accurately identifying cellular phenotypes and responses to external stimuli by combining molecular and biophysical information.

Endogenous protein "barcode" for data validation and normalization in quantitative MS analysis.

Science.gov (United States)

Lee, Wooram; Lazar, Iulia M

2014-07-01

Quantitative proteomic experiments with mass spectrometry detection are typically conducted by using stable isotope labeling and label-free quantitation approaches. Proteins with housekeeping functions and stable expression level such actin, tubulin, and glyceraldehyde-3-phosphate dehydrogenase are frequently used as endogenous controls. Recent studies have shown that the expression level of such common housekeeping proteins is, in fact, dependent on various factors such as cell type, cell cycle, or disease status and can change in response to a biochemical stimulation. The interference of such phenomena can, therefore, substantially compromise their use for data validation, alter the interpretation of results, and lead to erroneous conclusions. In this work, we advance the concept of a protein "barcode" for data normalization and validation in quantitative proteomic experiments. The barcode comprises a novel set of proteins that was generated from cell cycle experiments performed with MCF7, an estrogen receptor positive breast cancer cell line, and MCF10A, a nontumorigenic immortalized breast cell line. The protein set was selected from a list of ~3700 proteins identified in different cellular subfractions and cell cycle stages of MCF7/MCF10A cells, based on the stability of spectral count data generated with an LTQ ion trap mass spectrometer. A total of 11 proteins qualified as endogenous standards for the nuclear and 62 for the cytoplasmic barcode, respectively. The validation of the protein sets was performed with a complementary SKBR3/Her2+ cell line.

SILAC-Based Comparative Proteomic Analysis of Lysosomes from Mammalian Cells Using LC-MS/MS.

Science.gov (United States)

Thelen, Melanie; Winter, Dominic; Braulke, Thomas; Gieselmann, Volkmar

2017-01-01

Mass spectrometry-based proteomics of lysosomal proteins has led to significant advances in understanding lysosomal function and pathology. The ever-increasing sensitivity and resolution of mass spectrometry in combination with labeling procedures which allow comparative quantitative proteomics can be applied to shed more light on the steadily increasing range of lysosomal functions. In addition, investigation of alterations in lysosomal protein composition in the many lysosomal storage diseases may yield further insights into the molecular pathology of these disorders. Here, we describe a protocol which allows to determine quantitative differences in the lysosomal proteome of cells which are genetically and/or biochemically different or have been exposed to certain stimuli. The method is based on stable isotope labeling of amino acids in cell culture (SILAC). Cells are exposed to superparamagnetic iron oxide particles which are endocytosed and delivered to lysosomes. After homogenization of cells, intact lysosomes are rapidly enriched by passing the cell homogenates over a magnetic column. Lysosomes are eluted after withdrawal of the magnetic field and subjected to mass spectrometry.

Identification of Thioredoxin Disulfide Targets Using a Quantitative Proteomics Approach Based on Isotope-Coded Affinity Tags

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Maeda, Kenji

2008-01-01

Thioredoxin (Trx) is a ubiquitous protein disulfide reductase involved in a wide range of cellular redox processes. A large number of putative target proteins have been identified using proteomics approaches, but insight into target specificity at the molecular level is lacking since the reactivity...... of Trx toward individual disulfides has not been quantified. Here, a novel proteomics procedure is described for quantification of Trx-mediated target disulfide reduction based on thiol-specific differential labeling with the iodoacetamide-based isotope-coded affinity tag (ICAT) reagents. Briefly......, protein extract of embryos from germinated barley seeds was treated +/- Trx, and thiols released from target protein disulfides were irreversibly blocked with iodoacetamide. The remaining cysteine residues in the Trx-treated and the control (-Trx) samples were then chemically reduced and labeled...

Global analysis of the yeast osmotic stress response by quantitative proteomics

DEFF Research Database (Denmark)

Soufi, Boumediene; Kelstrup, C.D.; Stoehr, G.

2009-01-01

a comprehensive, quantitative, and time-resolved analysis using high-resolution mass spectrometry of phospho-proteome and proteome changes in response to osmotic stress in yeast. We identified 5534 unique phosphopeptide variants and 3383 yeast proteins. More than 15% of the detected phosphorylation site status...... changed more than two-fold within 5 minutes of treatment. Many of the corresponding phosphoproteins are involved in the early response to environmental stress. Surprisingly, we find that 158 regulated phosphorylation sites are potential substrates of basophilic kinases as opposed to the classical proline......-directed MAP kinase network implicated in stress response mechanisms such as p38 and HOG pathways. Proteome changes reveal an increase in abundance of more than one hundred proteins after 20 min of salt stress. Many of these are involved in the cellular response to increased osmolarity, which include proteins...

Cell-free protein synthesis: applications in proteomics and biotechnology.

Science.gov (United States)

He, Mingyue

2008-01-01

Protein production is one of the key steps in biotechnology and functional proteomics. Expression of proteins in heterologous hosts (such as in E. coli) is generally lengthy and costly. Cell-free protein synthesis is thus emerging as an attractive alternative. In addition to the simplicity and speed for protein production, cell-free expression allows generation of functional proteins that are difficult to produce by in vivo systems. Recent exploitation of cell-free systems enables novel development of technologies for rapid discovery of proteins with desirable properties from very large libraries. This article reviews the recent development in cell-free systems and their application in the large scale protein analysis.

Defining Diagnostic Biomarkers Using Shotgun Proteomics and MALDI-TOF Mass Spectrometry.

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Armengaud, Jean

2017-01-01

Whole-cell MALDI-TOF has become a robust and widely used tool to quickly identify any pathogen. In addition to being routinely used in hospitals, it is also useful for low cost dereplication in large scale screening procedures of new environmental isolates for environmental biotechnology or taxonomical applications. Here, I describe how specific biomarkers can be defined using shotgun proteomics and whole-cell MALDI-TOF mass spectrometry. Based on MALDI-TOF spectra recorded on a given set of pathogens with internal calibrants, m/z values of interest are extracted. The proteins which contribute to these peaks are deduced from label-free shotgun proteomics measurements carried out on the same sample. Quantitative information based on the spectral count approach allows ranking the most probable candidates. Proteogenomic approaches help to define whether these proteins give the same m/z values along the whole taxon under consideration or result in heterogeneous lists. These specific biomarkers nicely complement conventional profiling approaches and may help to better define groups of organisms, for example at the subspecies level.

A Probabilistic Framework for Peptide and Protein Quantification from Data-Dependent and Data-Independent LC-MS Proteomics Experiments

DEFF Research Database (Denmark)

Richardson, Katherine; Denny, R.; Hughes, C.

2012-01-01

A probability-based quantification framework is presented for the calculation of relative peptide and protein abundance in label-free and label-dependent LC-MS proteomics data. The results are accompanied by credible intervals and regulation probabilities. The algorithm takes into account data un...

Proteomic changes in response to crystal formation in Drosophila Malpighian tubules.

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Chung, Vera Y; Konietzny, Rebecca; Charles, Philip; Kessler, Benedikt; Fischer, Roman; Turney, Benjamin W

2016-04-02

Kidney stone disease is a major health burden with a complex and poorly understood pathophysiology. Drosophila Malpighian tubules have been shown to resemble human renal tubules in their physiological function. Herein, we have used Drosophila as a model to study the proteomic response to crystal formation induced by dietary manipulation in Malpighian tubules. Wild-type male flies were reared in parallel groups on standard medium supplemented with lithogenic agents: control, Sodium Oxalate (NaOx) and Ethylene Glycol (EG). Malpighian tubules were dissected after 2 weeks to visualize crystals with polarized light microscopy. The parallel group was dissected for protein extraction. A new method of Gel Assisted Sample Preparation (GASP) was used for protein extraction. Differentially abundant proteins (p<0.05) were identified by label-free quantitative proteomic analysis in flies fed with NaOx and EG diet compared with control. Their molecular functions were further screened for transmembrane ion transporter, calcium or zinc ion binder. Among these, 11 candidate proteins were shortlisted in NaOx diet and 16 proteins in EG diet. We concluded that GASP is a proteomic sample preparation method that can be applied to individual Drosophila Malpighian tubules. Our results may further increase the understanding of the pathophysiology of human kidney stone disease.

Responsive Hydrogels for Label-Free Signal Transduction within Biosensors

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Kamila Gawel

2010-04-01

Full Text Available Hydrogels have found wide application in biosensors due to their versatile nature. This family of materials is applied in biosensing either to increase the loading capacity compared to two-dimensional surfaces, or to support biospecific hydrogel swelling occurring subsequent to specific recognition of an analyte. This review focuses on various principles underpinning the design of biospecific hydrogels acting through various molecular mechanisms in transducing the recognition event of label-free analytes. Towards this end, we describe several promising hydrogel systems that when combined with the appropriate readout platform and quantitative approach could lead to future real-life applications.

Time-resolved quantitative proteome profiling of host-pathogen interactions: the response of Staphylococcus aureus RN1HG to internalisation by human airway epithelial cells.

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Schmidt, Frank; Scharf, Sandra S; Hildebrandt, Petra; Burian, Marc; Bernhardt, Jörg; Dhople, Vishnu; Kalinka, Julia; Gutjahr, Melanie; Hammer, Elke; Völker, Uwe

2010-08-01

Staphylococcus aureus is a versatile gram-positive pathogen that gains increasing importance due to the rapid spreading of resistances. Functional genomics technologies can provide new insights into the adaptational network of this bacterium and its response to environmental challenges. While functional genomics technologies, including proteomics, have been extensively used to study these phenomena in shake flask cultures, studies of bacteria from in vivo settings lack behind. Particularly for proteomics studies, the major bottleneck is the lack of sufficient proteomic coverage for low numbers of cells. In this study, we introduce a workflow that combines a pulse-chase stable isotope labelling by amino acids in cell culture approach with high capacity cell sorting, on-membrane digestion, and high-sensitivity MS to detect and quantitatively monitor several hundred S. aureus proteins from a few million internalised bacteria. This workflow has been used in a proof-of-principle experiment to reveal changes in levels of proteins with a function in protection against oxidative damage and adaptation of cell wall synthesis in strain RN1HG upon internalisation by S9 human bronchial epithelial cells.

Classification-based quantitative analysis of stable isotope labeling by amino acids in cell culture (SILAC) data.

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Kim, Seongho; Carruthers, Nicholas; Lee, Joohyoung; Chinni, Sreenivasa; Stemmer, Paul

2016-12-01

Stable isotope labeling by amino acids in cell culture (SILAC) is a practical and powerful approach for quantitative proteomic analysis. A key advantage of SILAC is the ability to simultaneously detect the isotopically labeled peptides in a single instrument run and so guarantee relative quantitation for a large number of peptides without introducing any variation caused by separate experiment. However, there are a few approaches available to assessing protein ratios and none of the existing algorithms pays considerable attention to the proteins having only one peptide hit. We introduce new quantitative approaches to dealing with SILAC protein-level summary using classification-based methodologies, such as Gaussian mixture models with EM algorithms and its Bayesian approach as well as K-means clustering. In addition, a new approach is developed using Gaussian mixture model and a stochastic, metaheuristic global optimization algorithm, particle swarm optimization (PSO), to avoid either a premature convergence or being stuck in a local optimum. Our simulation studies show that the newly developed PSO-based method performs the best among others in terms of F1 score and the proposed methods further demonstrate the ability of detecting potential markers through real SILAC experimental data. No matter how many peptide hits the protein has, the developed approach can be applicable, rescuing many proteins doomed to removal. Furthermore, no additional correction for multiple comparisons is necessary for the developed methods, enabling direct interpretation of the analysis outcomes. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Patterns of free amino acids in German convenience food products: marked mismatch between label information and composition.

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Hermanussen, M; Gonder, U; Jakobs, C; Stegemann, D; Hoffmann, G

2010-01-01

Free amino acids affect food palatability. As information on amino acids in frequently purchased pre-packaged food is virtually absent, we analyzed free amino acid patterns of 17 frequently purchased ready-to-serve convenience food products, and compared them with the information obtained from the respective food labels. Quantitative amino acid analysis was performed using ion-exchange chromatography. gamma-Aminobutyric acid (GABA) concentrations were verified using a stable isotope dilution gas chromatography/mass spectrometry (GC-MS) method. The patterns of free amino acids were compared with information obtained from food labels. An obvious mismatch between free amino acid patterns and food label information was detected. Even on considering that tomatoes and cereal proteins are naturally rich in glutamate, the concentrations of free glutamate outranged the natural concentration of this amino acid in several products, and strongly suggested artificial enrichment. Free glutamate was found to be elevated even in dishes that explicitly state 'no glutamate added'. Arginine was markedly elevated in lentils. Free cysteine was generally low, possibly reflecting thermal destruction of this amino acid during food processing. The meat and brain-specific dipeptide carnosine (CARN) was present in most meat-containing products. Some products did not contain detectable amounts of CARN in spite of meat content being claimed on the food labels. We detected GABA at concentrations that contribute significantly to the taste sensation. This investigation highlights a marked mismatch between food label information and food composition.

Proteome analysis of a hepatocyte-specific BIRC5 (survivin)-knockout mouse model during liver regeneration.

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Bracht, Thilo; Hagemann, Sascha; Loscha, Marius; Megger, Dominik A; Padden, Juliet; Eisenacher, Martin; Kuhlmann, Katja; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2014-06-06

The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5.

PCR-free quantitative detection of genetically modified organism from raw materials. An electrochemiluminescence-based bio bar code method.

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Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R

2008-05-15

A bio bar code assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio bar code assay requires lengthy experimental procedures including the preparation and release of bar code DNA probes from the target-nanoparticle complex and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio bar code assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2,2'-bipyridyl) ruthenium (TBR)-labeled bar code DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products.

Quantitative proteomic characterization of the lung extracellular matrix in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.

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Åhrman, Emma; Hallgren, Oskar; Malmström, Lars; Hedström, Ulf; Malmström, Anders; Bjermer, Leif; Zhou, Xiao-Hong; Westergren-Thorsson, Gunilla; Malmström, Johan

2018-03-01

Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues. We present a sequential tissue extraction strategy to determine changes in extractability of matrisome proteins in end-stage COPD and IPF compared to healthy control tissue. Extensive quantitative analysis of the proteome changes of the disease states revealed altered solubility of matrisome proteins involved in ECM regulators and cell-ECM communication. The results highlight disease-specific remodeling mechanisms associated with COPD and IPF. Copyright © 2018 Elsevier B.V. All rights reserved.

Quantitative Clinical Chemistry Proteomics (qCCP) using mass spectrometry: general characteristics and application.

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Lehmann, Sylvain; Hoofnagle, Andrew; Hochstrasser, Denis; Brede, Cato; Glueckmann, Matthias; Cocho, José A; Ceglarek, Uta; Lenz, Christof; Vialaret, Jérôme; Scherl, Alexander; Hirtz, Christophe

2013-05-01

Proteomics studies typically aim to exhaustively detect peptides/proteins in a given biological sample. Over the past decade, the number of publications using proteomics methodologies has exploded. This was made possible due to the availability of high-quality genomic data and many technological advances in the fields of microfluidics and mass spectrometry. Proteomics in biomedical research was initially used in 'functional' studies for the identification of proteins involved in pathophysiological processes, complexes and networks. Improved sensitivity of instrumentation facilitated the analysis of even more complex sample types, including human biological fluids. It is at that point the field of clinical proteomics was born, and its fundamental aim was the discovery and (ideally) validation of biomarkers for the diagnosis, prognosis, or therapeutic monitoring of disease. Eventually, it was recognized that the technologies used in clinical proteomics studies [particularly liquid chromatography-tandem mass spectrometry (LC-MS/MS)] could represent an alternative to classical immunochemical assays. Prior to deploying MS in the measurement of peptides/proteins in the clinical laboratory, it seems likely that traditional proteomics workflows and data management systems will need to adapt to the clinical environment and meet in vitro diagnostic (IVD) regulatory constraints. This defines a new field, as reviewed in this article, that we have termed quantitative Clinical Chemistry Proteomics (qCCP).

Comprehensive quantitative comparison of the membrane proteome, phosphoproteome, and sialiome of human embryonic and neural stem cells

DEFF Research Database (Denmark)

Melo-Braga, Marcella Nunes; Schulz, Melanie; Liu, Qiuyue

2014-01-01

Human embryonic stem cells (hESCs) can differentiate into neural stem cells (NSCs), which can further be differentiated into neurons and glia cells. Therefore, these cells have huge potential as source for treatment of neurological diseases. Membrane-associated proteins are very important......ESCs and NSCs as well as to investigate potential new markers for these two cell stages, we performed large-scale quantitative membrane-proteomic of hESCs and NSCs. This approach employed membrane purification followed by peptide dimethyl labeling and peptide enrichment to study the membrane subproteome as well...... in which 78% of phosphopeptides were identified with ≥99% confidence in site assignment and 1810 unique formerly sialylated N-linked glycopeptides. Several proteins were identified as significantly regulated in hESCs and NSC, including proteins involved in the early embryonic and neural development...

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

International Nuclear Information System (INIS)

Kasprzyk, P.G.; Cuttitta, F.; Avis, I.; Nakanishi, Y.; Treston, A.; Wong, H.; Walsh, J.H.; Mulshine, J.L.

1988-01-01

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones

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

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Mani D R

2011-08-01

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 http://www.hephy.at/user/fru/medea/.

ProteomicsDB.

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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

2018-01-04

ProteomicsDB (https://www.ProteomicsDB.org) 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.

Proteomics pipeline for biomarker discovery of laser capture microdissected breast cancer tissue

NARCIS (Netherlands)

N.Q. Liu (Ning Qing); R.B.H. Braakman (René); C. Stingl (Christoph); T.M. Luider (Theo); J.W.M. Martens (John); J.A. Foekens (John); A. Umar (Arzu)

2012-01-01

textabstractMass spectrometry (MS)-based label-free proteomics offers an unbiased approach to screen biomarkers related to disease progression and therapy-resistance of breast cancer on the global scale. However, multi-step sample preparation can introduce large variation in generated data, while

Proteome stability analysis of snap frozen, RNAlater preserved, and formalin-fixed paraffin-embedded human colon mucosal biopsies

DEFF Research Database (Denmark)

Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona

2016-01-01

Large repositories of well characterized RNAlater preserved samples and formalin-fixed, paraffin-embedded samples have been generated worldwide. However, the impact on the proteome of the preservation methods remain poorly described. Therefore, we analyzed the impact on the proteome of preserving...... throughput gel free quantitative proteomics. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002029....

Quantitative proteomic analysis of serum from pregnant women carrying a fetus with conotruncal heart defect using isobaric tags for relative and absolute quantitation (iTRAQ labeling.

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Ying Zhang

Full Text Available To identify differentially expressed proteins from serum of pregnant women carrying a conotruncal heart defects (CTD fetus, using proteomic analysis.The study was conducted using a nested case-control design. The 5473 maternal serum samples were collected at 14-18 weeks of gestation. The serum from 9 pregnant women carrying a CTD fetus, 10 with another CHD (ACHD fetus, and 11 with a normal fetus were selected from the above samples, and analyzed by using isobaric tags for relative and absolute quantitation (iTRAQ coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS. The differentially expressed proteins identified by iTRAQ were further validated with Western blot.A total of 105 unique proteins present in the three groups were identified, and relative expression data were obtained for 92 of them with high confidence by employing the iTRAQ-based experiments. The downregulation of gelsolin in maternal serum of fetus with CTD was further verified by Western blot.The identification of differentially expressed protein gelsolin in the serum of the pregnant women carrying a CTD fetus by using proteomic technology may be able to serve as a foundation to further explore the biomarker for detection of CTD fetus from the maternal serum.

Quantitative proteomic analysis of extracellular matrix extracted from mono- and dual-species biofilms of Fusobacterium nucleatum and Porphyromonas gingivalis.

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Mohammed, Marwan Mansoor Ali; Pettersen, Veronika Kuchařová; Nerland, Audun H; Wiker, Harald G; Bakken, Vidar

2017-04-01

The Gram-negative bacteria Fusobacterium nucleatum and Porphyromonas gingivalis are members of a complex dental biofilm associated with periodontal disease. In this study, we cultured F. nucleatum and P. gingivalis as mono- and dual-species biofilms, and analyzed the protein composition of the biofilms extracellular polymeric matrix (EPM) by high-resolution liquid chromatography-tandem mass spectrometry. Label-free quantitative proteomic analysis was used for identification of proteins and sequence-based functional characterization for their classification and prediction of possible roles in EPM. We identified 542, 93 and 280 proteins in the matrix of F. nucleatum, P. gingivalis, and the dual-species biofilm, respectively. Nearly 70% of all EPM proteins in the dual-species biofilm originated from F. nucleatum, and a majority of these were cytoplasmic proteins, suggesting an enhanced lysis of F. nucleatum cells. The proteomic analysis also indicated an interaction between the two species: 22 F. nucleatum proteins showed differential levels between the mono and dual-species EPMs, and 11 proteins (8 and 3 from F. nucleatum and P. gingivalis, respectively) were exclusively detected in the dual-species EPM. Oxidoreductases and chaperones were among the most abundant proteins identified in all three EPMs. The biofilm matrices in addition contained several known and hypothetical virulence proteins, which can mediate adhesion to the host cells and disintegration of the periodontal tissues. This study demonstrated that the biofilm matrix of two important periodontal pathogens consists of a multitude of proteins whose amounts and functionalities vary largely. Relatively high levels of several of the detected proteins might facilitate their potential use as targets for the inhibition of biofilm development. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative proteomic analysis of human lung tumor xenografts treated with the ectopic ATP synthase inhibitor citreoviridin.

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Yi-Hsuan Wu

Full Text Available ATP synthase is present on the plasma membrane of several types of cancer cells. Citreoviridin, an ATP synthase inhibitor, selectively suppresses the proliferation and growth of lung cancer without affecting normal cells. However, the global effects of targeting ectopic ATP synthase in vivo have not been well defined. In this study, we performed quantitative proteomic analysis using isobaric tags for relative and absolute quantitation (iTRAQ and provided a comprehensive insight into the complicated regulation by citreoviridin in a lung cancer xenograft model. With high reproducibility of the quantitation, we obtained quantitative proteomic profiling with 2,659 proteins identified. Bioinformatics analysis of the 141 differentially expressed proteins selected by their relative abundance revealed that citreoviridin induces alterations in the expression of glucose metabolism-related enzymes in lung cancer. The up-regulation of enzymes involved in gluconeogenesis and storage of glucose indicated that citreoviridin may reduce the glycolytic intermediates for macromolecule synthesis and inhibit cell proliferation. Using comprehensive proteomics, the results identify metabolic aspects that help explain the antitumorigenic effect of citreoviridin in lung cancer, which may lead to a better understanding of the links between metabolism and tumorigenesis in cancer therapy.

A proteomics approach to the identification of biomarkers for psoriasis utilising keratome biopsy

DEFF Research Database (Denmark)

Williamson, James C; Scheipers, Peter; Schwämmle, Veit

2013-01-01

a quantitative proteomics screen of four patients with psoriasis using stable isotope dimethyl labelling and identified over 50 proteins consistently altered in abundance in psoriasis lesional versus non-lesional skin. This includes several canonical psoriasis related proteins (e.g. S100A7 [Psoriasin] and FABP5...

PCR-free quantitative detection of genetically modified organism from raw materials – A novel electrochemiluminescence-based bio-barcode method

Science.gov (United States)

Zhu, Debin; Tang, Yabing; Xing, Da; Chen, Wei R.

2018-01-01

Bio-barcode assay based on oligonucleotide-modified gold nanoparticles (Au-NPs) provides a PCR-free method for quantitative detection of nucleic acid targets. However, the current bio-barcode assay requires lengthy experimental procedures including the preparation and release of barcode DNA probes from the target-nanoparticle complex, and immobilization and hybridization of the probes for quantification. Herein, we report a novel PCR-free electrochemiluminescence (ECL)-based bio-barcode assay for the quantitative detection of genetically modified organism (GMO) from raw materials. It consists of tris-(2’2’-bipyridyl) ruthenium (TBR)-labele barcode DNA, nucleic acid hybridization using Au-NPs and biotin-labeled probes, and selective capture of the hybridization complex by streptavidin-coated paramagnetic beads. The detection of target DNA is realized by direct measurement of ECL emission of TBR. It can quantitatively detect target nucleic acids with high speed and sensitivity. This method can be used to quantitatively detect GMO fragments from real GMO products. PMID:18386909

Multimodal label-free microscopy

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Nicolas Pavillon

2014-09-01

Full Text Available This paper reviews the different multimodal applications based on a large extent of label-free imaging modalities, ranging from linear to nonlinear optics, while also including spectroscopic measurements. We put specific emphasis on multimodal measurements going across the usual boundaries between imaging modalities, whereas most multimodal platforms combine techniques based on similar light interactions or similar hardware implementations. In this review, we limit the scope to focus on applications for biology such as live cells or tissues, since by their nature of being alive or fragile, we are often not free to take liberties with the image acquisition times and are forced to gather the maximum amount of information possible at one time. For such samples, imaging by a given label-free method usually presents a challenge in obtaining sufficient optical signal or is limited in terms of the types of observable targets. Multimodal imaging is then particularly attractive for these samples in order to maximize the amount of measured information. While multimodal imaging is always useful in the sense of acquiring additional information from additional modes, at times it is possible to attain information that could not be discovered using any single mode alone, which is the essence of the progress that is possible using a multimodal approach.

Proteomics assisted profiling of antimicrobial peptide signatures from black pepper (Piper nigrum L.).

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Umadevi, P; Soumya, M; George, Johnson K; Anandaraj, M

2018-05-01

Plant antimicrobial peptides are the interesting source of studies in defense response as they are essential components of innate immunity which exert rapid defense response. In spite of abundant reports on the isolation of antimicrobial peptides (AMPs) from many sources, the profile of AMPs expressed/identified from single crop species under certain stress/physiological condition is still unknown. This work describes the AMP signature profile of black pepper and their expression upon Phytophthora infection using label-free quantitative proteomics strategy. The differential expression of 24 AMPs suggests that a combinatorial strategy is working in the defense network. The 24 AMP signatures belonged to the cationic, anionic, cysteine-rich and cysteine-free group. As the first report on the possible involvement of AMP signature in Phytophthora infection, our results offer a platform for further study on regulation, evolutionary importance and exploitation of theses AMPs as next generation molecules against pathogens.

A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis.

Science.gov (United States)

Zhang, Xu; Liu, Qun; Zhou, Wei; Li, Ping; Alolga, Raphael N; Qi, Lian-Wen; Yin, Xiaojian

2018-03-30

Cordyceps sinensis has gained increasing attention due to its nutritional and medicinal properties. Herein, we employed label-free quantitative mass spectrometry to explore the proteome differences between naturally- and artificially-cultivated C. sinensis. A total of 22,829 peptides with confidence ≥95%, corresponding to 2541 protein groups were identified from the caterpillar bodies/stromata of 12 naturally- and artificially-cultivated samples of C. sinensis. Among them, 165 proteins showed significant differences between the samples of natural and artificial cultivation. These proteins were mainly involved in energy production/conversion, amino acid transport/metabolism, and transcription regulation. The proteomic results were confirmed by the identification of 4 significantly changed metabolites, thus, lysine, threonine, serine, and arginine via untargeted metabolomics. The change tendencies of these metabolites were partly in accordance with changes in abundance of the proteins, which was upstream of their synthetic pathways. In addition, the nutritional value in terms of the levels of nucleosides, nucleotides, and adenosine between the artificially- and naturally-cultivated samples was virtually same. These proteomic data will be useful for understanding the medicinal value of C. sinensis and serve as reference for its artificial cultivation. C. sinensis is a precious and valued medicinal product, the current basic proteome dataset would provide useful information to understand its development/infection processes as well as help to artificially cultivate it. This work would also provide basic proteome profile for further study of C. sinensis. Copyright © 2018. Published by Elsevier B.V.

Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count

Science.gov (United States)

Schmidt, Carla; Grønborg, Mads; Deckert, Jochen; Bessonov, Sergey; Conrad, Thomas; Lührmann, Reinhard; Urlaub, Henning

2014-01-01

The spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique. PMID:24448447

Application of targeted quantitative proteomics analysis in human cerebrospinal fluid using a liquid chromatography matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (LC MALDI TOF/TOF) platform.

Science.gov (United States)

Pan, Sheng; Rush, John; Peskind, Elaine R; Galasko, Douglas; Chung, Kathryn; Quinn, Joseph; Jankovic, Joseph; Leverenz, James B; Zabetian, Cyrus; Pan, Catherine; Wang, Yan; Oh, Jung Hun; Gao, Jean; Zhang, Jianpeng; Montine, Thomas; Zhang, Jing

2008-02-01

Targeted quantitative proteomics by mass spectrometry aims to selectively detect one or a panel of peptides/proteins in a complex sample and is particularly appealing for novel biomarker verification/validation because it does not require specific antibodies. Here, we demonstrated the application of targeted quantitative proteomics in searching, identifying, and quantifying selected peptides in human cerebrospinal spinal fluid (CSF) using a matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer (MALDI TOF/TOF)-based platform. The approach involved two major components: the use of isotopic-labeled synthetic peptides as references for targeted identification and quantification and a highly selective mass spectrometric analysis based on the unique characteristics of the MALDI instrument. The platform provides high confidence for targeted peptide detection in a complex system and can potentially be developed into a high-throughput system. Using the liquid chromatography (LC) MALDI TOF/TOF platform and the complementary identification strategy, we were able to selectively identify and quantify a panel of targeted peptides in the whole proteome of CSF without prior depletion of abundant proteins. The effectiveness and robustness of the approach associated with different sample complexity, sample preparation strategies, as well as mass spectrometric quantification were evaluated. Other issues related to chromatography separation and the feasibility for high-throughput analysis were also discussed. Finally, we applied targeted quantitative proteomics to analyze a subset of previously identified candidate markers in CSF samples of patients with Parkinson's disease (PD) at different stages and Alzheimer's disease (AD) along with normal controls.

Label-free probing of genes by time-domain terahertz sensing

International Nuclear Information System (INIS)

Bolivar, P Haring; Brucherseifer, M; Nagel, M; Kurz, H; Bosserhoff, A; Buettner, R

2002-01-01

A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed

Label-free probing of genes by time-domain terahertz sensing

Energy Technology Data Exchange (ETDEWEB)

Bolivar, P Haring [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Brucherseifer, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Nagel, M [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Kurz, H [Institut fuer Halbleitertechnik, RWTH Aachen, Sommerfeldstr. 24, D-52056 Aachen (Germany); Bosserhoff, A [Institut fuer Pathologie, Universitaet Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg (Germany); Buettner, R [Institut fuer Pathologie, Universitaetsklinikum Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn (Germany)

2002-11-07

A label-free sensing approach for the label-free characterization of genetic material with terahertz (THz) electromagnetic waves is presented. Time-resolved THz analysis of polynucleotides demonstrates a strong dependence of the complex refractive index of DNA molecules in the THz frequency range on their hybridization state. By monitoring THz signals one can thus infer the binding state (hybridized or denatured) of oligo- and polynucleotides, enabling the label-free determination the genetic composition of unknown DNA sequences. A broadband experimental proof-of-principle in a free-space analytic configuration, as well as a higher-sensitivity approach using integrated THz sensors reaching femtomol detection levels and demonstrating the capability to detect single-base mutations, are presented. The potential application for next generation high-throughput label-free genetic analytic systems is discussed.

Label free imaging of cell-substrate contacts by holographic total internal reflection microscopy.

Science.gov (United States)

Mandracchia, Biagio; Gennari, Oriella; Marchesano, Valentina; Paturzo, Melania; Ferraro, Pietro

2017-09-01

The study of cell adhesion contacts is pivotal to understand cell mechanics and interaction at substrates or chemical and physical stimuli. We designed and built a HoloTIR microscope for label-free quantitative phase imaging of total internal reflection. Here we show for the first time that HoloTIR is a good choice for label-free study of focal contacts and of cell/substrate interaction as its sensitivity is enhanced in comparison with standard TIR microscopy. Finally, the simplicity of implementation and relative low cost, due to the requirement of less optical components, make HoloTIR a reasonable alternative, or even an addition, to TIRF microscopy for mapping cell/substratum topography. As a proof of concept, we studied the formation of focal contacts of fibroblasts on three substrates with different levels of affinity for cell adhesion. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Enhancing cognate target elution efficiency in gel-free chemical proteomics

Directory of Open Access Journals (Sweden)

Branka Radic-Sarikas

2015-12-01

Full Text Available Gel-free liquid chromatography mass spectrometry coupled to chemical proteomics is a powerful approach for characterizing cellular target profiles of small molecules. We have previously described a fast and efficient elution protocol; however, altered target profiles were observed. We hypothesised that elution conditions critically impact the effectiveness of disrupting drug-protein interactions. Thus, a number of elution conditions were systematically assessed with the aim of improving the recovery of all classes of proteins whilst maintaining compatibility with immunoblotting procedures. A double elution with formic acid combined with urea emerged as the most efficient and generically applicable elution method for chemical proteomics

Label-free LC-MS analysis of HER2+ breast cancer cell line response to HER2 inhibitor treatment.

Science.gov (United States)

Di Luca, Alessio; Henry, Michael; Meleady, Paula; O'Connor, Robert

2015-08-04

Human epidermal growth-factor receptor (HER)-2 is overexpressed in 25 % of breast-cancers and is associated with an aggressive form of the disease with significantly shortened disease free and overall survival. In recent years, the use of HER2-targeted therapies, monoclonal-antibodies and small molecule tyrosine-kinase inhibitors has significantly improved the clinical outcome for HER2-positive breast-cancer patients. However, only a fraction of HER2-amplified patients will respond to therapy and the use of these treatments is often limited by tumour drug insensitivity or resistance and drug toxicities. Currently there is no way to identify likely responders or rational combinations with the potential to improve HER2-focussed treatment outcome. In order to further understand the molecular mechanisms of treatment-response with HER2-inhibitors, we used a highly-optimised and reproducible quantitative label-free LC-MS strategy to characterize the proteomes of HER2-overexpressing breast-cancer cell-lines (SKBR3, BT474 and HCC1954) in response to drug-treatment with HER2-inhibitors (lapatinib, neratinib or afatinib). Following 12 ours treatment with different HER2-inhibitors in the BT474 cell-line; compared to the untreated cells, 16 proteins changed significantly in abundance following lapatinib treatment (1 μM), 21 proteins changed significantly following neratinib treatment (150 nM) and 38 proteins changed significantly following afatinib treatment (150 nM). Whereas following 24 hours treatment with neratinib (200 nM) 46 proteins changed significantly in abundance in the HCC1954 cell-line and 23 proteins in the SKBR3 cell-line compared to the untreated cells. Analysing the data we found that, proteins like trifunctional-enzyme subunit-alpha, mitochondrial; heterogeneous nuclear ribonucleoprotein-R and lamina-associated polypeptide 2, isoform alpha were up-regulated whereas heat shock cognate 71 kDa protein was down-regulated in 3 or more comparisons. This proteomic

Mining the human urine proteome for monitoring renal transplant injury

Energy Technology Data Exchange (ETDEWEB)

Sigdel, Tara K.; Gao, Yuqian; He, Jintang; Wang, Anyou; Nicora, Carrie D.; Fillmore, Thomas L.; Shi, Tujin; Webb-Robertson, Bobbie-Jo; Smith, Richard D.; Qian, Wei-Jun; Salvatierra, Oscar; Camp, David G.; Sarwal, Minnie M.

2016-06-01

The human urinary proteome reflects systemic and inherent renal injury perturbations and can be analyzed to harness specific biomarkers for different kidney transplant injury states. 396 unique urine samples were collected contemporaneously with an allograft biopsy from 396 unique kidney transplant recipients. Centralized, blinded histology on the graft was used to classify matched urine samples into categories of acute rejection (AR), chronic allograft nephropathy (CAN), BK virus nephritis (BKVN), and stable graft (STA). Liquid chromatography–mass spectrometry (LC-MS) based proteomics using iTRAQ based discovery (n=108) and global label-free LC-MS analyses of individual samples (n=137) for quantitative proteome assessment were used in the discovery step. Selected reaction monitoring (SRM) was applied to identify and validate minimal urine protein/peptide biomarkers to accurately segregate organ injury causation and pathology on unique urine samples (n=151). A total of 958 proteins were initially quantified by iTRAQ, 87% of which were also identified among 1574 urine proteins detected in LC-MS validation. 103 urine proteins were significantly (p<0.05) perturbed in injury and enriched for humoral immunity, complement activation, and lymphocyte trafficking. A set of 131 peptides corresponding to 78 proteins were assessed by SRM for their significance in an independent sample cohort. A minimal set of 35 peptides mapping to 33 proteins, were modeled to segregate different injury groups (AUC =93% for AR, 99% for CAN, 83% for BKVN). Urinary proteome discovery and targeted validation identified urine protein fingerprints for non-invasive differentiation of kidney transplant injuries, thus opening the door for personalized immune risk assessment and therapy.

Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders

OpenAIRE

Zhang, Lijun; Jia, Xiaofang; Jin, Jun-O; Lu, Hongzhou; Tan, Zhimi

2017-01-01

Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as wel...

Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics

DEFF Research Database (Denmark)

Ong, S.E.; Blagoev, B.; Kratchmarova, I.

2002-01-01

Quantitative proteomics has traditionally been performed by two-dimensional gel electrophoresis, but recently, mass spectrometric methods based on stable isotope quantitation have shown great promise for the simultaneous and automated identification and quantitation of complex protein mixtures. H...

Optimized Clinical Use of RNALater and FFPE Samples for Quantitative Proteomics

DEFF Research Database (Denmark)

Bennike, Tue Bjerg; Kastaniegaard, Kenneth; Padurariu, Simona

2015-01-01

Introduction and Objectives The availability of patient samples is essential for clinical proteomic research. Biobanks worldwide store mainly samples stabilized in RNAlater as well as formalin-fixed and paraffin embedded (FFPE) biopsies. Biobank material is a potential source for clinical...... we compare to FFPE and frozen samples being the control. Methods From the sigmoideum of two healthy participants’ twenty-four biopsies were extracted using endoscopy. The biopsies was stabilized either by being directly frozen, RNAlater, FFPE or incubated for 30 min at room temperature prior to FFPE...... information. Conclusion We have demonstrated that quantitative proteome analysis and pathway mapping of samples stabilized in RNAlater as well as by FFPE is feasible with minimal impact on the quality of protein quantification and post-translational modifications....

Quantitative Proteomics Analysis Identifies Mitochondria as Therapeutic Targets of Multidrug-Resistance in Ovarian Cancer

Science.gov (United States)

Chen, Xiulan; Wei, Shasha; Ma, Ying; Lu, Jie; Niu, Gang; Xue, Yanhong; Chen, Xiaoyuan; Yang, Fuquan

2014-01-01

Doxorubicin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors. However, resistance to this anticancer drug is a major obstacle to the effective treatment of tumors. As mitochondria play important roles in cell life and death, we anticipate that mitochondria may be related to drug resistance. Here, stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomic strategy was applied to compare mitochondrial protein expression in doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI_ADR/RES cells. A total of 2085 proteins were quantified, of which 122 proteins displayed significant changes in the NCI_ADR/RES cells. These proteins participated in a variety of cell processes including cell apoptosis, substance metabolism, transport, detoxification and drug metabolism. Then qRT-PCR and western blot were applied to validate the differentially expressed proteins quantified by SILAC. Further functional studies with RNAi demonstrated TOP1MT, a mitochondrial protein participated in DNA repair, was involved in doxorubicin resistance in NCI_ADR/RES cells. Besides the proteomic study, electron microscopy and fluorescence analysis also observed that mitochondrial morphology and localization were greatly altered in NCI_ADR/RES cells. Mitochondrial membrane potential was also decreased in NCI_ADR/RES cells. All these results indicate that mitochondrial function is impaired in doxorubicin-resistant cells and mitochondria play an important role in doxorubicin resistance. This research provides some new information about doxorubicin resistance, indicating that mitochondria could be therapeutic targets of doxorubicin resistance in ovarian cancer cells. PMID:25285166

Using label-free screening technology to improve efficiency in drug discovery.

Science.gov (United States)

Halai, Reena; Cooper, Matthew A

2012-02-01

Screening assays have traditionally utilized reporter labels to quantify biological responses relevant to the disease state of interest. However, there are limitations associated with the use of labels that may be overcome with temporal measurements possible with label-free. This review comprises general and system-specific information from literature searches using PubMed, published books and the authors' personal experience. This review highlights the label-free approaches in the context of various applications. The authors also note technical issues relevant to the development of label-free assays and their application to HTS. The limitations associated with the use of transfected cell lines and the use of label-based assays are gradually being realized. As such, greater emphasis is being placed on label-free biophysical techniques using native cell lines. The introduction of 96- and 384-well plate label-free systems is helping to broker a wider acceptance of these approaches in high-throughput screening. However, potential users of the technologies remain skeptical, primarily because the physical basis of the signals generated, and their contextual relevance to cell biology and signal transduction, has not been fully elucidated. Until this is done, these new technology platforms are more likely to complement, rather than replace, traditional screening platforms.

Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

Directory of Open Access Journals (Sweden)

Theresa Wan Chen Yap

2014-01-01

Full Text Available Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

Growth phase-dependent proteomes of the Malaysian isolated Lactococcus lactis dairy strain M4 using label-free qualitative shotgun proteomics analysis.

Science.gov (United States)

Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Rahim, Raha Abdul; Mahadi, Nor Muhammad; Illias, Rosli Md; Murad, Abdul Munir Abdul

2014-01-01

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MS(E)) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications.

Growth Phase-Dependent Proteomes of the Malaysian Isolated Lactococcus lactis Dairy Strain M4 Using Label-Free Qualitative Shotgun Proteomics Analysis

Science.gov (United States)

Yap, Theresa Wan Chen; Rabu, Amir; Abu Bakar, Farah Diba; Abdul Rahim, Raha; Mahadi, Nor Muhammad; Illias, Rosli Md.

2014-01-01

Lactococcus lactis is the most studied mesophilic fermentative lactic acid bacterium. It is used extensively in the food industry and plays a pivotal role as a cell factory and also as vaccine delivery platforms. The proteome of the Malaysian isolated L. lactis M4 dairy strain, obtained from the milk of locally bred cows, was studied to elucidate the physiological changes occurring between the growth phases of this bacterium. In this study, ultraperformance liquid chromatography nanoflow electrospray ionization tandem mass spectrometry (UPLC- nano-ESI-MSE) approach was used for qualitative proteomic analysis. A total of 100 and 121 proteins were identified from the midexponential and early stationary growth phases, respectively, of the L. lactis strain M4. During the exponential phase, the most important reaction was the generation of sufficient energy, whereas, in the early stationary phase, the metabolic energy pathways decreased and the biosynthesis of proteins became more important. Thus, the metabolism of the cells shifted from energy production in the exponential phase to the synthesis of macromolecules in the stationary phase. The resultant proteomes are essential in providing an improved view of the cellular machinery of L. lactis during the transition of growth phases and hence provide insight into various biotechnological applications. PMID:24982972

Potential protein biomarkers for burning mouth syndrome discovered by quantitative proteomics.

Science.gov (United States)

Ji, Eoon Hye; Diep, Cynthia; Liu, Tong; Li, Hong; Merrill, Robert; Messadi, Diana; Hu, Shen

2017-01-01

Burning mouth syndrome (BMS) is a chronic pain disorder characterized by severe burning sensation in normal looking oral mucosa. Diagnosis of BMS remains to be a challenge to oral healthcare professionals because the method for definite diagnosis is still uncertain. In this study, a quantitative saliva proteomic analysis was performed in order to identify target proteins in BMS patients' saliva that may be used as biomarkers for simple, non-invasive detection of the disease. By using isobaric tags for relative and absolute quantitation labeling and liquid chromatography-tandem mass spectrometry to quantify 1130 saliva proteins between BMS patients and healthy control subjects, we found that 50 proteins were significantly changed in the BMS patients when compared to the healthy control subjects ( p ≤ 0.05, 39 up-regulated and 11 down-regulated). Four candidates, alpha-enolase, interleukin-18 (IL-18), kallikrein-13 (KLK13), and cathepsin G, were selected for further validation. Based on enzyme-linked immunosorbent assay measurements, three potential biomarkers, alpha-enolase, IL-18, and KLK13, were successfully validated. The fold changes for alpha-enolase, IL-18, and KLK13 were determined as 3.6, 2.9, and 2.2 (burning mouth syndrome vs. control), and corresponding receiver operating characteristic values were determined as 0.78, 0.83, and 0.68, respectively. Our findings indicate that testing of the identified protein biomarkers in saliva might be a valuable clinical tool for BMS detection. Further validation studies of the identified biomarkers or additional candidate biomarkers are needed to achieve a multi-marker prediction model for improved detection of BMS with high sensitivity and specificity.

The colon mucosa is a potential site for the initial triggering of Rheumatoid Arthritis

DEFF Research Database (Denmark)

Bennike, Tue Bjerg; Ellingsen, Torkell Juulsgaad; Glerup, Henning

2017-01-01

extracted by colonoscopy and analyzed by label-free quantitative proteomics, enabling the quantitation of 5366 proteins. The abundance of dihydrofolate reductase (DHFR) was statistically significantly increased in RA-patient biopsies compared with controls and correlated with the administered dosage...

Surveying alignment-free features for Ortholog detection in related yeast proteomes by using supervised big data classifiers.

Science.gov (United States)

Galpert, Deborah; Fernández, Alberto; Herrera, Francisco; Antunes, Agostinho; Molina-Ruiz, Reinaldo; Agüero-Chapin, Guillermin

2018-05-03

The development of new ortholog detection algorithms and the improvement of existing ones are of major importance in functional genomics. We have previously introduced a successful supervised pairwise ortholog classification approach implemented in a big data platform that considered several pairwise protein features and the low ortholog pair ratios found between two annotated proteomes (Galpert, D et al., BioMed Research International, 2015). The supervised models were built and tested using a Saccharomycete yeast benchmark dataset proposed by Salichos and Rokas (2011). Despite several pairwise protein features being combined in a supervised big data approach; they all, to some extent were alignment-based features and the proposed algorithms were evaluated on a unique test set. Here, we aim to evaluate the impact of alignment-free features on the performance of supervised models implemented in the Spark big data platform for pairwise ortholog detection in several related yeast proteomes. The Spark Random Forest and Decision Trees with oversampling and undersampling techniques, and built with only alignment-based similarity measures or combined with several alignment-free pairwise protein features showed the highest classification performance for ortholog detection in three yeast proteome pairs. Although such supervised approaches outperformed traditional methods, there were no significant differences between the exclusive use of alignment-based similarity measures and their combination with alignment-free features, even within the twilight zone of the studied proteomes. Just when alignment-based and alignment-free features were combined in Spark Decision Trees with imbalance management, a higher success rate (98.71%) within the twilight zone could be achieved for a yeast proteome pair that underwent a whole genome duplication. The feature selection study showed that alignment-based features were top-ranked for the best classifiers while the runners-up were

Quantitation of thrombogenicity of hemodialyzer with technetium-99m and indium-111 labeled platelets

International Nuclear Information System (INIS)

Dewanjee, M.K.; Kapadvanjwala, M.; Ruzius, K.; Serafini, A.N.; Zilleruelo, G.E.; Sfakianakis, G.N.

1993-01-01

The platelet thrombogenicity of a hemodialyzer was quantified with 99m Tc- and 111 In-labeled platelets. The platelets collected from blood of Beagle dogs, Yorkshire pigs and humans were labeled with 111 In-tropolone (detergent-free) and 99m Tc-HMPAO. Hemodialysis was performed with a hollow-fiber dialyzer (HFD) in a flow-loop, the temperature maintained at 37 o C, with flow-rates of 7, 150 and 270 mL/min; after dialysis, HFD radioactivity was measured with an ionization chamber and imaged with a γ-camera. The dynamic processes of thrombosis and embolization from the hemodialyzer resulted in large standard deviations around the mean values of the adherent thrombus. In spite of this, the quantitation of comparative thrombogenicity with 111 In-and 99m Tc-labeled platelets suggests that both radionuclides could be used for measurement of device-induced thrombogenicity and estimation of prosthesis-induced thrombogenicity. (author)

Platelets Proteomic Profiles of Acute Ischemic Stroke Patients.

Directory of Open Access Journals (Sweden)

Ozge Cevik

Full Text Available Platelets play a crucial role in the pathogenesis of stroke and antiplatelet agents exist for its treatment and prevention. Through the use of LC-MS based protein expression profiling, platelets from stroke patients were analyzed and then correlated with the proteomic analyses results in the context of this disease. This study was based on patients who post ischemic stroke were admitted to hospital and had venous blood drawn within 24 hrs of the incidence. Label-free protein expression analyses of the platelets' tryptic digest was performed in triplicate on a UPLC-ESI-qTOF-MS/MS system and ProteinLynx Global Server (v2.5, Waters was used for tandem mass data extraction. The peptide sequences were searched against the reviewed homo sapiens database (www.uniprot.org and the quantitation of protein variation was achieved through Progenesis LC-MS software (V4.0, Nonlinear Dynamics. These Label-free differential proteomics analysis of platelets ensured that 500 proteins were identified and 83 of these proteins were found to be statistically significant. The differentially expressed proteins are involved in various processes such as inflammatory response, cellular movement, immune cell trafficking, cell-to-cell signaling and interaction, hematological system development and function and nucleic acid metabolism. The expressions of myeloperoxidase, arachidonate 12-Lipoxygenase and histidine-rich glycoprotein are involved in cellular metabolic processes, crk-like protein and ras homolog gene family member A involved in cell signaling with vitronectin, thrombospondin 1, Integrin alpha 2b, and integrin beta 3 involved in cell adhesion. Apolipoprotein H, immunoglobulin heavy constant gamma 1 and immunoglobulin heavy constant gamma 3 are involved in structural, apolipoprotein A-I, and alpha-1-microglobulin/bikunin precursor is involved in transport, complement component 3 and clusterin is involved in immunity proteins as has been discussed. Our data provides

In-depth Proteomics Characterization of Embryogenesis of the Honey Bee Worker (Apis mellifera ligustica) *

Science.gov (United States)

Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

2014-01-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24–48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48–72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified

Adaptation of a Commonly Used, Chemically Defined Medium for Human Embryonic Stem Cells to Stable Isotope Labeling with Amino Acids in Cell Culture

DEFF Research Database (Denmark)

Liberski, A. R.; Al-Noubi, M. N.; Rahman, Z. H.

2013-01-01

Metabolic labeling with stable isotopes is a prominent technique for comparative quantitative proteomics, and stable isotope labeling with amino acids in cell culture (SILAC) is the most commonly used approach. SILAC is, however, traditionally limited to simple tissue culture regimens and only ra...

The goat (Capra hircus) mammary gland secretory tissue proteome as influenced by weight loss: A study using label free proteomics

Science.gov (United States)

Seasonal weight loss (SWL) is a significant limitation to animal production. Breeds that have evolved in harsh climates have acquired tolerance to SWL through selection. Herein, labelfree proteomics was used to characterize the effects of SWL in two goat breeds with different levels of adaptation to...

Proteomic cornerstones of hematopoietic stem cell differentiation

DEFF Research Database (Denmark)

Klimmeck, Daniel; Hansson, Jenny; Raffel, Simon

2012-01-01

Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors...... which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem....../progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5,000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical...

Halobacterium salinarum NRC-1 PeptideAtlas: strategies for targeted proteomics

Science.gov (United States)

Van, Phu T.; Schmid, Amy K.; King, Nichole L.; Kaur, Amardeep; Pan, Min; Whitehead, Kenia; Koide, Tie; Facciotti, Marc T.; Goo, Young-Ah; Deutsch, Eric W.; Reiss, David J.; Mallick, Parag; Baliga, Nitin S.

2009-01-01

The relatively small numbers of proteins and fewer possible posttranslational modifications in microbes provides a unique opportunity to comprehensively characterize their dynamic proteomes. We have constructed a Peptide Atlas (PA) for 62.7% of the predicted proteome of the extremely halophilic archaeon Halobacterium salinarum NRC-1 by compiling approximately 636,000 tandem mass spectra from 497 mass spectrometry runs in 88 experiments. Analysis of the PA with respect to biophysical properties of constituent peptides, functional properties of parent proteins of detected peptides, and performance of different mass spectrometry approaches has helped highlight plausible strategies for improving proteome coverage and selecting signature peptides for targeted proteomics. Notably, discovery of a significant correlation between absolute abundances of mRNAs and proteins has helped identify low abundance of proteins as the major limitation in peptide detection. Furthermore we have discovered that iTRAQ labeling for quantitative proteomic analysis introduces a significant bias in peptide detection by mass spectrometry. Therefore, despite identifying at least one proteotypic peptide for almost all proteins in the PA, a context-dependent selection of proteotypic peptides appears to be the most effective approach for targeted proteomics. PMID:18652504

Recent 5-year Findings and Technological Advances in the Proteomic Study of HIV-associated Disorders.

Science.gov (United States)

Zhang, Lijun; Jia, Xiaofang; Jin, Jun-O; Lu, Hongzhou; Tan, Zhimi

2017-04-01

Human immunodeficiency virus-1 (HIV-1) mainly relies on host factors to complete its life cycle. Hence, it is very important to identify HIV-regulated host proteins. Proteomics is an excellent technique for this purpose because of its high throughput and sensitivity. In this review, we summarized current technological advances in proteomics, including general isobaric tags for relative and absolute quantitation (iTRAQ) and stable isotope labeling by amino acids in cell culture (SILAC), as well as subcellular proteomics and investigation of posttranslational modifications. Furthermore, we reviewed the applications of proteomics in the discovery of HIV-related diseases and HIV infection mechanisms. Proteins identified by proteomic studies might offer new avenues for the diagnosis and treatment of HIV infection and the related diseases. Copyright © 2017 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Tissue-based quantitative proteome analysis of human hepatocellular carcinoma using tandem mass tags.

Science.gov (United States)

Megger, Dominik Andre; Rosowski, Kristin; Ahrens, Maike; Bracht, Thilo; Eisenacher, Martin; Schlaak, Jörg F; Weber, Frank; Hoffmann, Andreas-Claudius; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

2017-03-01

Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics. Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed. RAB1A was verified to be a potent biomarker candidate for HCC.

Golgi enrichment and proteomic analysis of developing Pinus radiata xylem by free-flow electrophoresis.

Directory of Open Access Journals (Sweden)

Harriet T Parsons

Full Text Available Our understanding of the contribution of Golgi proteins to cell wall and wood formation in any woody plant species is limited. Currently, little Golgi proteomics data exists for wood-forming tissues. In this study, we attempted to address this issue by generating and analyzing Golgi-enriched membrane preparations from developing xylem of compression wood from the conifer Pinus radiata. Developing xylem samples from 3-year-old pine trees were harvested for this purpose at a time of active growth and subjected to a combination of density centrifugation followed by free flow electrophoresis, a surface charge separation technique used in the enrichment of Golgi membranes. This combination of techniques was successful in achieving an approximately 200-fold increase in the activity of the Golgi marker galactan synthase and represents a significant improvement for proteomic analyses of the Golgi from conifers. A total of thirty known Golgi proteins were identified by mass spectrometry including glycosyltransferases from gene families involved in glucomannan and glucuronoxylan biosynthesis. The free flow electrophoresis fractions of enriched Golgi were highly abundant in structural proteins (actin and tubulin indicating a role for the cytoskeleton during compression wood formation. The mass spectrometry proteomics data associated with this study have been deposited to the ProteomeXchange with identifier PXD000557.

Quantitative proteomics reveals divergent responses in Apis mellifera worker and drone pupae to parasitization by Varroa destructor.

Science.gov (United States)

Surlis, Carla; Carolan, James C; Coffey, Mary; Kavanagh, Kevin

Varroa destructor is a haemophagous ectoparasite of honeybees and is considered a major causal agent of colony losses in Europe and North America. Although originating in Eastern Asia where it parasitizes Apis cerana, it has shifted hosts to the western honeybee Apis mellifera on which it has a greater deleterious effect on the individual and colony level. To investigate this important host-parasite interaction and to determine whether Varroa causes different effects on different castes we conducted a label free quantitative proteomic analysis of Varroa-parasitized and non-parasitized drone and worker Apis mellifera pupae. 1195 proteins were identified in total, of which 202 and 250 were differentially abundant in parasitized drone and worker pupae, respectively. Both parasitized drone and worker pupae displayed reduced abundance in proteins associated with the cuticle, lipid transport and innate immunity. Proteins involved in metabolic processes were more abundant in both parasitized castes although the response in workers was more pronounced. A number of caste specific responses were observed including differential abundance of numerous cytoskeletal and muscle proteins, which were of higher abundance in parasitized drones in comparison to parasitized workers. Proteins involved in fatty acid and carbohydrate metabolism were more abundant in parasitized workers as were a large number of ribosomal proteins highlighting either potentially divergent responses to Varroa or a different strategy by the mite when parasitizing the different castes. This data improves our understanding of this interaction and may provide a basis for future studies into improvements to therapy and control of Varroasis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Extensive mass spectrometry proteomics data of Persicaria minor herb upon methyl jasmonate treatment

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Wan Mohd Aizat

2018-02-01

Full Text Available Proteomics is often hindered by the lack of protein sequence database particularly for non-model species such as Persicaria minor herbs. An integrative approach called proteomics informed by transcriptomics is possible [1], in which translated transcriptome sequence database is used as the protein sequence database. In this current study, the proteome profile were profiled using SWATH-MS technology complemented with documented transcriptome profiling [2], the first such report in this tropical herb. The plant was also elicited using a phytohormone, methyl jasmonate (MeJA and protein changes were elucidated using label-free quantification of SWATH-MS to understand the role of such signal molecule in this herbal species. The mass spectrometry proteomics data was deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD005749. This data article refers to the article entitled “Proteomics (SWATH-MS-informed by transcriptomics approach of Persicaria minor leaves upon methyl jasmonate elicitation” [3].

Label-free and selective nonlinear fiber-optical biosensing

DEFF Research Database (Denmark)

Ott, Johan Raunkjær; Heuck, Mikkel; Agger, Christian

2008-01-01

We demonstrate that the inherent nonlinearity of a microstructured optical fiber (MOF) may be used to achieve label-free selective biosensing, thereby eliminating the need for post-processing of the fiber. This first nonlinear biosensor utilizes a change in the modulational instability (MI) gain...... for optimizing the sensitivity. The nonlinear sensor shows a sensitivity of around 10.4nm/nm, defined as the shift in resonance wavelength per nm biolayer, which is a factor of 7.5 higher than the hitherto only demonstrated label-free MOF biosensor....

Quantitative proteomic studies in resistance mechanisms of Eimeria tenella against polyether ionophores.

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Thabet, Ahmed; Honscha, Walther; Daugschies, Arwid; Bangoura, Berit

2017-05-01

Polyether ionophores are widely used to treat and control coccidiosis in chickens. Widespread use of anticoccidials resulted in worldwide resistance. Mechanisms of resistance development and expansion are complex and poorly understood. Relative proteomic quantification using LC-MS/MS was used to compare sensitive reference strains (Ref-1, Ref-2) with putatively resistant and moderately sensitive field strains (FS-R, FS-mS) of Eimeria tenella after isotopic labelling with tandem mass tags (TMT). Ninety-seven proteins were identified, and 25 of them were regulated. Actin was significantly upregulated in resistant strains in comparison with their sensitive counterparts. On the other hand, microneme protein (MIC4) was downregulated in resistant strains. Optimization of labelling E. tenella sporozoites by TMT might identify further proteins that play a role in the obvious complex mechanism leading to resistance against Monensin.

Comparative Proteomic Profiling of Mycobacterium bovis and BCG Vaccine Strains

KAUST Repository

Gao, Ge

2013-09-01

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.

Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

NARCIS (Netherlands)

Lundby, Alicia; Rossin, Elizabeth J.; Steffensen, Annette B.; Acha, Moshe Ray; Newton-Cheh, Christopher; Pfeufer, Arne; Lyneh, Stacey N.; Olesen, Soren-Peter; Brunak, Soren; Ellinor, Patrick T.; Jukema, J. Wouter; Trompet, Stella; Ford, Ian; Macfarlane, Peter W.; Krijthe, Bouwe P.; Hofman, Albert; Uitterlinden, Andre G.; Stricker, Bruno H.; Nathoe, Hendrik M.; Spiering, Wilko; Daly, Mark J.; Asselbergs, Ikea W.; van der Harst, Pim; Milan, David J.; de Bakker, Paul I. W.; Lage, Kasper; Olsen, Jesper V.

Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes

Stable isotope N-phosphoryl amino acids labeling for quantitative profiling of amine-containing metabolites using liquid chromatography mass spectrometry.

Science.gov (United States)

Zhang, Shanshan; Shi, Jinwen; Shan, Changkai; Huang, Chengting; Wu, Yile; Ding, Rong; Xue, Yuhua; Liu, Wen; Zhou, Qiang; Zhao, Yufen; Xu, Pengxiang; Gao, Xiang

2017-07-25

Stable isotope chemical labeling liquid chromatography-mass spectrometry (LC-MS) is a powerful strategy for comprehensive metabolomics profiling, which can improve metabolites coverage and quantitative information for exploration of metabolic regulation in complex biological systems. In the current work, a novel stable isotope N-phosphoryl amino acids labeling strategy (SIPAL) has been successful developed for quantitative profiling of amine-containing metabolites in urine based on organic phosphorus chemistry. Two isotopic reagents, 16 O 2 - and 18 O 2 -N-diisopropyl phosphoryl l-alanine N-hydroxysuccinimide esters ( 16 O/ 18 O-DIPP-L-Ala-NHS), were firstly synthesized in high yields for labeling the amine-containing metabolites. The performance of SIPAL strategy was tested by analyzing standard samples including 20 l-amino acids, 10 d-amino acids and small peptides by using LC-MS. We observed highly efficient and selective labeling for SIPAL strategy within 15 min in a one-pot derivatization reaction under aqueous reaction conditions. The introduction of a neutral phosphate group at N-terminus can increase the proton affinity and overall hydrophobicity of targeted metabolites, leading to the better ionization efficiency in electrospray ionization processes and chromatographic separations of hydrophilic metabolites on reversed-phase column. Furthermore, the chiral metabolites, such as d-amino acids, could be converted to diastereomers after SIPAL and successfully separated on regular reversed-phase column. The chirality of labeled enantiomers can be determined by using different detection methods such as 31 P NMR, UV, and MS, demonstrating the potential application of SIPAL strategy. In addition, absolute quantification of chiral metabolites in biological samples can be easily achieved by using SIPAL strategy. For this purpose, urine samples collected from a healthy volunteer were analyzed by using LC-ESI-Orbitrap MS. Over 300 pairs of different amine

Evaluation of Normalization Methods on GeLC-MS/MS Label-Free Spectral Counting Data to Correct for Variation during Proteomic Workflows

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Gokce, Emine; Shuford, Christopher M.; Franck, William L.; Dean, Ralph A.; Muddiman, David C.

2011-12-01

Normalization of spectral counts (SpCs) in label-free shotgun proteomic approaches is important to achieve reliable relative quantification. Three different SpC normalization methods, total spectral count (TSpC) normalization, normalized spectral abundance factor (NSAF) normalization, and normalization to selected proteins (NSP) were evaluated based on their ability to correct for day-to-day variation between gel-based sample preparation and chromatographic performance. Three spectral counting data sets obtained from the same biological conidia sample of the rice blast fungus Magnaporthe oryzae were analyzed by 1D gel and liquid chromatography-tandem mass spectrometry (GeLC-MS/MS). Equine myoglobin and chicken ovalbumin were spiked into the protein extracts prior to 1D-SDS- PAGE as internal protein standards for NSP. The correlation between SpCs of the same proteins across the different data sets was investigated. We report that TSpC normalization and NSAF normalization yielded almost ideal slopes of unity for normalized SpC versus average normalized SpC plots, while NSP did not afford effective corrections of the unnormalized data. Furthermore, when utilizing TSpC normalization prior to relative protein quantification, t-testing and fold-change revealed the cutoff limits for determining real biological change to be a function of the absolute number of SpCs. For instance, we observed the variance decreased as the number of SpCs increased, which resulted in a higher propensity for detecting statistically significant, yet artificial, change for highly abundant proteins. Thus, we suggest applying higher confidence level and lower fold-change cutoffs for proteins with higher SpCs, rather than using a single criterion for the entire data set. By choosing appropriate cutoff values to maintain a constant false positive rate across different protein levels (i.e., SpC levels), it is expected this will reduce the overall false negative rate, particularly for proteins with

Proteomic evidences for rex regulation of metabolism in toxin-producing Bacillus cereus ATCC 14579.

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Sabrina Laouami

Full Text Available The facultative anaerobe, Bacillus cereus, causes diarrheal diseases in humans. Its ability to deal with oxygen availability is recognized to be critical for pathogenesis. The B. cereus genome comprises a gene encoding a protein with high similarities to the redox regulator, Rex, which is a central regulator of anaerobic metabolism in Bacillus subtilis and other Gram-positive bacteria. Here, we showed that B. cereus rex is monocistronic and down-regulated in the absence of oxygen. The protein encoded by rex is an authentic Rex transcriptional factor since its DNA binding activity depends on the NADH/NAD+ ratio. Rex deletion compromised the ability of B. cereus to cope with external oxidative stress under anaerobiosis while increasing B. cereus resistance against such stress under aerobiosis. The deletion of rex affects anaerobic fermentative and aerobic respiratory metabolism of B. cereus by decreasing and increasing, respectively, the carbon flux through the NADH-recycling lactate pathway. We compared both the cellular proteome and exoproteome of the wild-type and Δrex cells using a high throughput shotgun label-free quantitation approach and identified proteins that are under control of Rex-mediated regulation. Proteomics data have been deposited to the ProteomeXchange with identifier PXD000886. The data suggest that Rex regulates both the cross-talk between metabolic pathways that produce NADH and NADPH and toxinogenesis, especially in oxic conditions.

Optimized protein extraction for quantitative proteomics of yeasts.

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Tobias von der Haar

2007-10-01

Full Text Available The absolute quantification of intracellular protein levels is technically demanding, but has recently become more prominent because novel approaches like systems biology and metabolic control analysis require knowledge of these parameters. Current protocols for the extraction of proteins from yeast cells are likely to introduce artifacts into quantification procedures because of incomplete or selective extraction.We have developed a novel procedure for protein extraction from S. cerevisiae based on chemical lysis and simultaneous solubilization in SDS and urea, which can extract the great majority of proteins to apparent completeness. The procedure can be used for different Saccharomyces yeast species and varying growth conditions, is suitable for high-throughput extraction in a 96-well format, and the resulting extracts can easily be post-processed for use in non-SDS compatible procedures like 2D gel electrophoresis.An improved method for quantitative protein extraction has been developed that removes some of the sources of artefacts in quantitative proteomics experiments, while at the same time allowing novel types of applications.

Quantitative proteomic analysis of ibuprofen-degrading Patulibacter sp. strain I11

DEFF Research Database (Denmark)

Almeida, Barbara; Kjeldal, Henrik; Lolas, Ihab Bishara Yousef

2013-01-01

was identified and quantified by gel based shotgun-proteomics. In total 251 unique proteins were quantitated using this approach. Biological process and pathway analysis indicated a number of proteins that were up-regulated in response to active degradation of ibuprofen, some of them are known to be involved...... in the degradation of aromatic compounds. Data analysis revealed that several of these proteins are likely involved in ibuprofen degradation by Patulibacter sp. strain I11.......Ibuprofen is the third most consumed pharmaceutical drug in the world. Several isolates have been shown to degrade ibuprofen, but very little is known about the biochemistry of this process. This study investigates the degradation of ibuprofen by Patulibacter sp. strain I11 by quantitative...

Quantitative Proteomics Identifies Activation of Hallmark Pathways of Cancer in Patient Melanoma.

Science.gov (United States)

Byrum, Stephanie D; Larson, Signe K; Avaritt, Nathan L; Moreland, Linley E; Mackintosh, Samuel G; Cheung, Wang L; Tackett, Alan J

2013-03-01

Molecular pathways regulating melanoma initiation and progression are potential targets of therapeutic development for this aggressive cancer. Identification and molecular analysis of these pathways in patients has been primarily restricted to targeted studies on individual proteins. Here, we report the most comprehensive analysis of formalin-fixed paraffin-embedded human melanoma tissues using quantitative proteomics. From 61 patient samples, we identified 171 proteins varying in abundance among benign nevi, primary melanoma, and metastatic melanoma. Seventy-three percent of these proteins were validated by immunohistochemistry staining of malignant melanoma tissues from the Human Protein Atlas database. Our results reveal that molecular pathways involved with tumor cell proliferation, motility, and apoptosis are mis-regulated in melanoma. These data provide the most comprehensive proteome resource on patient melanoma and reveal insight into the molecular mechanisms driving melanoma progression.

Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

KAUST Repository

Chandramouli, Kondethimmanahalli

2013-03-01

Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

Transcriptome and quantitative proteome analysis reveals molecular processes associated with larval metamorphosis in the polychaete pseudopolydora vexillosa

KAUST Repository

Chandramouli, Kondethimmanahalli; Sun, Jin; Mok, FloraSy; Liu, Lingli; Qiu, Jianwen; Ravasi, Timothy; Qian, Peiyuan

2013-01-01

Larval growth of the polychaete worm Pseudopolydora vexillosa involves the formation of segment-specific structures. When larvae attain competency to settle, they discard swimming chaetae and secrete mucus. The larvae build tubes around themselves and metamorphose into benthic juveniles. Understanding the molecular processes, which regulate this complex and unique transition, remains a major challenge because of the limited molecular information available. To improve this situation, we conducted high-throughput RNA sequencing and quantitative proteome analysis of the larval stages of P. vexillosa. Based on gene ontology (GO) analysis, transcripts related to cellular and metabolic processes, binding, and catalytic activities were highly represented during larval-adult transition. Mitogen-activated protein kinase (MAPK), calcium-signaling, Wnt/β-catenin, and notch signaling metabolic pathways were enriched in transcriptome data. Quantitative proteomics identified 107 differentially expressed proteins in three distinct larval stages. Fourteen and 53 proteins exhibited specific differential expression during competency and metamorphosis, respectively. Dramatic up-regulation of proteins involved in signaling, metabolism, and cytoskeleton functions were found during the larval-juvenile transition. Several proteins involved in cell signaling, cytoskeleton and metabolism were up-regulated, whereas proteins related to transcription and oxidative phosphorylation were down-regulated during competency. The integration of high-throughput RNA sequencing and quantitative proteomics allowed a global scale analysis of larval transcripts/proteins associated molecular processes in the metamorphosis of polychaete worms. Further, transcriptomic and proteomic insights provide a new direction to understand the fundamental mechanisms that regulate larval metamorphosis in polychaetes. © 2013 American Chemical Society.

iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus.

Science.gov (United States)

Gao, Kun; Deng, Xiang-Yuan; Shang, Meng-Ke; Qin, Guang-Xing; Hou, Cheng-Xiang; Guo, Xi-Jie

2017-01-30

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) specifically infects the epithelial cells in the midgut of silkworm and causes them to death, which negatively affects the sericulture industry. In order to determine the midgut response at the protein levels to the virus infection, differential proteomes of the silkworm midgut responsive to BmCPV infection were identified with isobaric tags for relative and absolute quantitation (iTRAQ) labeling followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). 193, 408, 189 differentially expressed proteins (DEPs) were reliably quantified by iTRAQ analysis in the midgut of BmCPV-infected and control larvae at 24, 48, 72h post infection (hpi) respectively. KEGG enrichment analysis showed that Oxidative phosphorylation, amyotrophic lateral sclerosis, Toll-like receptor signaling pathway, steroid hormone biosynthesis were the significant pathways (Q value≤0.05) both at 24 and 48hpi. qRT-PCR was used to further verify gene transcription of 30 DEPs from iTRAQ, showing that the regulations of 24 genes at the transcript level were consistent with those at the proteomic level. Moreover, the cluster analysis of the three time groups showed that there were seven co-regulated DEPs including BGIBMGA002620-PA, which was a putative p62/sequestosome-1 protein in silkworm. It was upregulated at both the mRNA level and the proteomic level and may play an important role in regulating the autophagy and apoptosis (especially apoptosis) induced by BmCPV infection. This was the first report using an iTRAQ approach to analyze proteomes of the silkworm midgut against BmCPV infection, which contributes to understanding the defense mechanisms of silkworm midgut to virus infection. The domesticated silkworm, Bombyx mori, is renowned for silk production as well as being a traditional lepidopteron model insect served as a subject for morphological, genetic, physiological, and developmental studies. Bombyx mori cytoplasmic polyhedrosis

Proteome-wide dataset supporting functional study of tyrosine kinases in breast cancer

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Nicos Angelopoulos

2016-06-01

Full Text Available Tyrosine kinases (TKs play an essential role in regulating various cellular activities and dysregulation of TK signaling contributes to oncogenesis. However, less than half of the TKs have been thoroughly studied. Through a combined use of RNAi and stable isotope labeling with amino acids in cell culture (SILAC-based quantitative proteomics, a global functional proteomic landscape of TKs in breast cancer was recently revealed highlighting a comprehensive and highly integrated signaling network regulated by TKs (Stebbing et al., 2015 [1]. We collate the enormous amount of the proteomic data in an open access platform, providing a valuable resource for studying the function of TKs in cancer and benefiting the science community. Here we present a detailed description related to this study (Stebbing et al., 2015 [1] and the raw data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the identifier http://www.ebi.ac.uk/pride/archive/projects/PXD002065.

Proteomic Profiling of the Pituitary Gland in Studies of Psychiatric Disorders.

Science.gov (United States)

Krishnamurthy, Divya; Rahmoune, Hassan; Guest, Paul C

2017-01-01

Psychiatric disorders have been associated with perturbations of the hypothalamic-pituitary-adrenal axis. Therefore, proteomic studies of the pituitary gland have the potential to provide new insights into the underlying pathways affected in these conditions as well as identify new biomarkers or targets for use in developing improved medications. This chapter describes a protocol for preparation of pituitary protein extracts followed by characterization of the pituitary proteome by label-free liquid chromatography-tandem mass spectrometry in expression mode (LC-MS E ). The main focus was on establishing a method for identifying the major pituitary hormones and accessory proteins as many of these have already been implicated in psychiatric diseases.

Quantitative proteomics reveals the central changes of wheat in response to powdery mildew.

Science.gov (United States)

Fu, Ying; Zhang, Hong; Mandal, Siddikun Nabi; Wang, Changyou; Chen, Chunhuan; Ji, Wanquan

2016-01-01

Powdery mildew (Pm), caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most important crop diseases, causing severe economic losses to wheat production worldwide. However, there are few reports about the proteomic response to Bgt infection in resistant wheat. Hence, quantitative proteomic analysis of N9134, a resistant wheat line, was performed to explore the molecular mechanism of wheat in defense against Bgt. Comparing the leaf proteins of Bgt-inoculated N9134 with that of mock-inoculated controls, a total of 2182 protein-species were quantified by iTRAQ at 24, 48 and 72h postinoculation (hpi) with Bgt, of which 394 showed differential accumulation. These differentially accumulated protein-species (DAPs) mainly included pathogenesis-related (PR) polypeptides, oxidative stress responsive proteins and components involved in primary metabolic pathways. KEGG enrichment analysis showed that phenylpropanoid biosynthesis, phenylalanine metabolism and photosynthesis-antenna proteins were the key pathways in response to Bgt infection. InterProScan 5 and the Gibbs Motif Sampler cluster 394 DAPs into eight conserved motifs, which shared leucine repeats and histidine sites in the sequence motifs. Moreover, eight separate protein-protein interaction (PPI) networks were predicted from STRING database. This study provides a powerful platform for further exploration of the molecular mechanism underlying resistant wheat responding to Bgt. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a destructive pathogenic disease in wheat-producing regions worldwide, resulting in severe yield reductions. Although many resistant wheat varieties have been cultivated, there are few reports about the proteomic response to Bgt infection in resistant wheat. Therefore, an iTRAQ-based quantitative proteomic analysis of a resistant wheat line (N9134) in response to Bgt infection has been performed. This paper provides new insights into the underlying molecular

Gel-free proteomics reveal potential biomarkers of priming-induced salt tolerance in durum wheat.

Science.gov (United States)

Fercha, Azzedine; Capriotti, Anna Laura; Caruso, Giuseppe; Cavaliere, Chiara; Gherroucha, Hocine; Samperi, Roberto; Stampachiacchiere, Serena; Lagana, Aldo

2013-10-08

Seed priming has been successfully demonstrated to be an efficient method to improve crop productivity under stressful conditions. As a first step toward better understanding of the mechanisms underlying the priming-induced salt stress tolerance in durum wheat, and to overcome the limitations of the gel-based approach, a comparative gel-free proteomic analysis was conducted with durum wheat seed samples of varying vigor as generated by hydro- and ascorbate-priming treatments. Results indicate that hydro-priming was accompanied by significant changes of 72 proteins, most of which are involved in proteolysis, protein synthesis, metabolism and disease/defense response. Ascorbate-priming was, however, accompanied by significant changes of 83 proteins, which are mainly involved in protein metabolism, antioxidant protection, repair processes and, interestingly, in methionine-related metabolism. The present study provides new information for understanding how 'priming-memory' invokes seed stress tolerance. The current work describes the first study in which gel-free shotgun proteomics were used to investigate the metabolic seed protein fraction in durum wheat. A combined approach of protein fractionation, hydrogel nanoparticle enrichment technique, and gel-free shotgun proteomic analysis allowed us to identify over 380 proteins exhibiting greater molecular weight diversity (ranging from 7 to 258kDa). Accordingly, we propose that this approach could be useful to acquire a wider perspective and a better understanding of the seed proteome. In the present work, we employed this method to investigate the potential biomarkers of priming-induced salt tolerance in durum wheat. In this way, we identified several previously unrecognized proteins which were never been reported before, particularly for the ascorbate-priming treatment. These findings could provide new avenues for improving crop productivity, particularly under unfavorable environmental conditions. © 2013.

Quantitative Proteomic Analysis of Hepatic Tissue of T2DM Rhesus Macaque

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Tingfu Du

2017-01-01

Full Text Available Type 2 diabetes mellitus (T2DM is a metabolic disorder that severely affects human health, but the pathogenesis of the disease remains unknown. The high-fat/high-sucrose diets combined with streptozotocin- (STZ- induced nonhuman primate animal model of diabetes are a valuable research source of T2DM. Here, we present a study of a STZ rhesus macaque model of T2DM that utilizes quantitative iTRAQ-based proteomic method. We compared the protein profiles in the liver of STZ-treated macaques as well as age-matched healthy controls. We identified 171 proteins differentially expressed in the STZ-treated groups, about 70 of which were documented as diabetes-related gene in previous studies. Pathway analyses indicated that the biological functions of differentially expressed proteins were related to glycolysis/gluconeogenesis, fatty acid metabolism, complements, and coagulation cascades. Expression change in tryptophan metabolism pathway was also found in this study which may be associations with diabetes. This study is the first to explore genome-wide protein expression in hepatic tissue of diabetes macaque model using HPLC-Q-TOF/MS technology. In addition to providing potential T2DM biomarkers, this quantitative proteomic study may also shed insights regarding the molecular pathogenesis of T2DM.

NHS-based Tandem Mass Tagging of Proteins at the Level of Whole Cells: A Critical Evaluation in Comparison to Conventional TMT-Labeling Approaches for Quantitative Proteome Analysis.

Science.gov (United States)

Megger, Dominik A; Pott, Leona L; Rosowski, Kristin; Zülch, Birgit; Tautges, Stephanie; Bracht, Thilo; Sitek, Barbara

2017-01-01

Tandem mass tags (TMT) are usually introduced at the levels of isolated proteins or peptides. Here, for the first time, we report the labeling of whole cells and a critical evaluation of its performance in comparison to conventional labeling approaches. The obtained results indicated that TMT protein labeling using intact cells is generally possible, if it is coupled to a subsequent enrichment using anti-TMT antibody. The quantitative results were similar to those obtained after labeling of isolated proteins and both were found to be slightly complementary to peptide labeling. Furthermore, when using NHS-based TMT, no specificity towards cell surface proteins was observed in the case of cell labeling. In summary, the conducted study revealed first evidence for the general possibility of TMT cell labeling and highlighted limitations of NHS-based labeling reagents. Future studies should therefore focus on the synthesis and investigation of membrane impermeable TMTs to increase specificity towards cell surface proteins.

Quality evaluation of LC-MS/MS-based E. coli H antigen typing (MS-H) through label-free quantitative data analysis in a clinical sample setup.

Science.gov (United States)

Cheng, Keding; Sloan, Angela; McCorrister, Stuart; Peterson, Lorea; Chui, Huixia; Drebot, Mike; Nadon, Celine; Knox, J David; Wang, Gehua

2014-12-01

The need for rapid and accurate H typing is evident during Escherichia coli outbreak situations. This study explores the transition of MS-H, a method originally developed for rapid H antigen typing of E. coli using LC-MS/MS of flagella digest of reference strains and some clinical strains, to E. coli isolates in clinical scenario through quantitative analysis and method validation. Motile and nonmotile strains were examined in batches to simulate clinical sample scenario. Various LC-MS/MS batch run procedures and MS-H typing rules were compared and summarized through quantitative analysis of MS-H data output for a standard method development. Label-free quantitative data analysis of MS-H typing was proven very useful for examining the quality of MS-H result and the effects of some sample carryovers from motile E. coli isolates. Based on this, a refined procedure and protein identification rule specific for clinical MS-H typing was established and validated. With LC-MS/MS batch run procedure and database search parameter unique for E. coli MS-H typing, the standard procedure maintained high accuracy and specificity in clinical situations, and its potential to be used in a clinical setting was clearly established. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Proteomic characterization of EL4 lymphoma-derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase-6 during tumor cell death in vivo.

Science.gov (United States)

Kramer, David A; Eldeeb, Mohamed A; Wuest, Melinda; Mercer, John; Fahlman, Richard P

2017-06-01

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in-depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label-free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase-3 and an upregulation of caspase-6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Analysis of proteomes released from in vitro cultured eight Clostridium difficile PCR ribotypes revealed specific expression in PCR ribotypes 027 and 176 confirming their genetic relatedness and clinical importance at the proteomic level

Czech Academy of Sciences Publication Activity Database

Dresler, J.; Krůtová, M.; Fučíková, A.; Klimentová, J.; Hrůzová, V.; Ďuráčová, M.; Houdková, K.; Salovská, B.; Matějková, J.; Hubálek, Martin; Pajer, P.; Píša, L.; Nyč, O.

2017-01-01

Roč. 9, Aug 14 (2017), č. článku 45. ISSN 1757-4749 Institutional support: RVO:61388963 Keywords : Clostridium difficile * label-free quantification * proteome * PCR ribotype 027 * PCR ribotype 176 Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology Impact factor: 2.756, year: 2016 https://gutpathogens.biomedcentral.com/articles/10.1186/s13099-017-0194-9

PANDA-view: An easy-to-use tool for statistical analysis and visualization of quantitative proteomics data.

Science.gov (United States)

Chang, Cheng; Xu, Kaikun; Guo, Chaoping; Wang, Jinxia; Yan, Qi; Zhang, Jian; He, Fuchu; Zhu, Yunping

2018-05-22

Compared with the numerous software tools developed for identification and quantification of -omics data, there remains a lack of suitable tools for both downstream analysis and data visualization. To help researchers better understand the biological meanings in their -omics data, we present an easy-to-use tool, named PANDA-view, for both statistical analysis and visualization of quantitative proteomics data and other -omics data. PANDA-view contains various kinds of analysis methods such as normalization, missing value imputation, statistical tests, clustering and principal component analysis, as well as the most commonly-used data visualization methods including an interactive volcano plot. Additionally, it provides user-friendly interfaces for protein-peptide-spectrum representation of the quantitative proteomics data. PANDA-view is freely available at https://sourceforge.net/projects/panda-view/. 1987ccpacer@163.com and zhuyunping@gmail.com. Supplementary data are available at Bioinformatics online.

In-depth proteomics characterization of embryogenesis of the honey bee worker (Apis mellifera ligustica).

Science.gov (United States)

Fang, Yu; Feng, Mao; Han, Bin; Lu, Xiaoshan; Ramadan, Haitham; Li, Jianke

2014-09-01

Identifying proteome changes of honey bee embryogenesis is of prime importance for unraveling the molecular mechanisms that they underlie. However, many proteomic changes during the embryonic period are not well characterized. We analyzed the proteomic alterations over the complete time course of honey bee worker embryogenesis at 24, 48, and 72 h of age, using mass spectrometry-based proteomics, label-free quantitation, and bioinformatics. Of the 1460 proteins identified the embryo of all three ages, the core proteome (proteins shared by the embryos of all three ages, accounting for 40%) was mainly involved in protein synthesis, metabolic energy, development, and molecular transporter, which indicates their centrality in driving embryogenesis. However, embryos at different developmental stages have their own specific proteome and pathway signatures to coordinate and modulate developmental events. The young embryos (proteins related to nutrition storage and nucleic acid metabolism may correlate with the cell proliferation occurring at this stage. The middle aged embryos (24-48 h) enhanced expression of proteins associated with cell cycle control, transporters, antioxidant activity, and the cytoskeleton suggest their roles to support rudimentary organogenesis. Among these proteins, the biological pathways of aminoacyl-tRNA biosynthesis, β-alanine metabolism, and protein export are intensively activated in the embryos of middle age. The old embryos (48-72 h) elevated expression of proteins implicated in fatty acid metabolism and morphogenesis indicate their functionality for the formation and development of organs and dorsal closure, in which the biological pathways of fatty acid metabolism and RNA transport are highly activated. These findings add novel understanding to the molecular details of honey bee embryogenesis, in which the programmed activation of the proteome matches with the physiological transition observed during embryogenesis. The identified biological

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

Science.gov (United States)

Trinkle-Mulcahy, Laura; Boulon, Séverine; Lam, Yun Wah; Urcia, Roby; Boisvert, François-Michel; Vandermoere, Franck; Morrice, Nick A.; Swift, Sam; Rothbauer, Ulrich; Leonhardt, Heinrich; Lamond, Angus

2008-01-01

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments. PMID:18936248

Stable isotopic labeling-based quantitative targeted glycomics (i-QTaG).

Science.gov (United States)

Kim, Kyoung-Jin; Kim, Yoon-Woo; Kim, Yun-Gon; Park, Hae-Min; Jin, Jang Mi; Hwan Kim, Young; Yang, Yung-Hun; Kyu Lee, Jun; Chung, Junho; Lee, Sun-Gu; Saghatelian, Alan

2015-01-01

Mass spectrometry (MS) analysis combined with stable isotopic labeling is a promising method for the relative quantification of aberrant glycosylation in diseases and disorders. We developed a stable isotopic labeling-based quantitative targeted glycomics (i-QTaG) technique for the comparative and quantitative analysis of total N-glycans using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). We established the analytical procedure with the chemical derivatizations (i.e., sialic acid neutralization and stable isotopic labeling) of N-glycans using a model glycoprotein (bovine fetuin). Moreover, the i-QTaG using MALDI-TOF MS was evaluated with various molar ratios (1:1, 1:2, 1:5) of (13) C6 /(12) C6 -2-aminobenzoic acid-labeled glycans from normal human serum. Finally, this method was applied to direct comparison of the total N-glycan profiles between normal human sera (n = 8) and prostate cancer patient sera (n = 17). The intensities of the N-glycan peaks from i-QTaG method showed a good linearity (R(2) > 0.99) with the amount of the bovine fetuin glycoproteins. The ratios of relative intensity between the isotopically 2-AA labeled N-glycans were close to the theoretical molar ratios (1:1, 1:2, 1:5). We also demonstrated that the up-regulation of the Lewis antigen (~82%) in sera from prostate cancer patients. In this proof-of-concept study, we demonstrated that the i-QTaG method, which enables to achieve a reliable comparative quantitation of total N-glycans via MALDI-TOF MS analysis, has the potential to diagnose and monitor alterations in glycosylation associated with disease states or biotherapeutics. © 2015 American Institute of Chemical Engineers.

Proteome Analysis of Human Arterial Tissue Discloses Associations Between the Vascular Content of Small Leucine-Rich Repeat Proteoglycans and Pulse Wave Velocity

DEFF Research Database (Denmark)

Lyck Hansen, Maria; Beck, Hans Christian; Irmukhamedov, Akhmadjon

2015-01-01

OBJECTIVES: We hypothesized that arterial stiffness is associated with changes in the arterial protein profile, particularly of extracellular matrix components. We aimed at determining differentially expressed proteins by quantitative proteome analysis in arterial tissue from patients with differ......OBJECTIVES: We hypothesized that arterial stiffness is associated with changes in the arterial protein profile, particularly of extracellular matrix components. We aimed at determining differentially expressed proteins by quantitative proteome analysis in arterial tissue from patients...... with different degrees of arterial stiffness. APPROACH AND RESULTS: Arterial stiffness, assessed by carotid-femoral pulse wave velocity (PWV), central blood pressure and augmentation index by pulse wave analysis were measured the day before surgery in a group of patients undergoing coronary artery bypass...... grafting. Protein extracts of well-defined, homogenous, nonatherosclerotic individual samples of the left mammary artery from 10 of these patients with high PWV and 9 with low PWV were compared by quantitative proteome analysis, using tandem mass tag labeling and nano-liquid chromatography mass...

iTRAQ-Based Quantitative Proteomics of Developing and Ripening Muscadine Grape Berry

Science.gov (United States)

Kambiranda, Devaiah; Katam, Ramesh; Basha, Sheikh M.; Siebert, Shalom

2014-01-01

Grapes are among the widely cultivated fruit crops in the world. Grape berries like other nonclimacteric fruits undergo a complex set of dynamic, physical, physiological, and biochemical changes during ripening. Muscadine grapes are widely cultivated in the southern United States for fresh fruit and wine. To date, changes in the metabolites composition of muscadine grapes have been well documented; however, the molecular changes during berry development and ripening are not fully known. The aim of this study was to investigate changes in the berry proteome during ripening in muscadine grape cv. Noble. Isobaric tags for relative and absolute quantification (iTRAQ) MS/MS was used to detect statistically significant changes in the berry proteome. A total of 674 proteins were detected, and 76 were differentially expressed across four time points in muscadine berry. Proteins obtained were further analyzed to provide information about its potential functions during ripening. Several proteins involved in abiotic and biotic stimuli and sucrose and hexose metabolism were upregulated during berry ripening. Quantitative real-time PCR analysis validated the protein expression results for nine proteins. Identification of vicilin-like antimicrobial peptides indicates additional disease tolerance proteins are present in muscadines for berry protection during ripening. The results provide new information for characterization and understanding muscadine berry proteome and grape ripening. PMID:24251720

Multidimensional electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) for quantitative analysis of the proteome and phosphoproteome in clinical and biomedical research.

Science.gov (United States)

Loroch, Stefan; Schommartz, Tim; Brune, Wolfram; Zahedi, René Peiman; Sickmann, Albert

2015-05-01

Quantitative proteomics and phosphoproteomics have become key disciplines in understanding cellular processes. Fundamental research can be done using cell culture providing researchers with virtually infinite sample amounts. In contrast, clinical, pre-clinical and biomedical research is often restricted to minute sample amounts and requires an efficient analysis with only micrograms of protein. To address this issue, we generated a highly sensitive workflow for combined LC-MS-based quantitative proteomics and phosphoproteomics by refining an ERLIC-based 2D phosphoproteomics workflow into an ERLIC-based 3D workflow covering the global proteome as well. The resulting 3D strategy was successfully used for an in-depth quantitative analysis of both, the proteome and the phosphoproteome of murine cytomegalovirus-infected mouse fibroblasts, a model system for host cell manipulation by a virus. In a 2-plex SILAC experiment with 150 μg of a tryptic digest per condition, the 3D strategy enabled the quantification of ~75% more proteins and even ~134% more peptides compared to the 2D strategy. Additionally, we could quantify ~50% more phosphoproteins by non-phosphorylated peptides, concurrently yielding insights into changes on the levels of protein expression and phosphorylation. Beside its sensitivity, our novel three-dimensional ERLIC-strategy has the potential for semi-automated sample processing rendering it a suitable future perspective for clinical, pre-clinical and biomedical research. Copyright © 2015. Published by Elsevier B.V.

Label-free tissue scanner for colorectal cancer screening

Science.gov (United States)

Kandel, Mikhail E.; Sridharan, Shamira; Liang, Jon; Luo, Zelun; Han, Kevin; Macias, Virgilia; Shah, Anish; Patel, Roshan; Tangella, Krishnarao; Kajdacsy-Balla, Andre; Guzman, Grace; Popescu, Gabriel

2017-06-01

The current practice of surgical pathology relies on external contrast agents to reveal tissue architecture, which is then qualitatively examined by a trained pathologist. The diagnosis is based on the comparison with standardized empirical, qualitative assessments of limited objectivity. We propose an approach to pathology based on interferometric imaging of "unstained" biopsies, which provides unique capabilities for quantitative diagnosis and automation. We developed a label-free tissue scanner based on "quantitative phase imaging," which maps out optical path length at each point in the field of view and, thus, yields images that are sensitive to the "nanoscale" tissue architecture. Unlike analysis of stained tissue, which is qualitative in nature and affected by color balance, staining strength and imaging conditions, optical path length measurements are intrinsically quantitative, i.e., images can be compared across different instruments and clinical sites. These critical features allow us to automate the diagnosis process. We paired our interferometric optical system with highly parallelized, dedicated software algorithms for data acquisition, allowing us to image at a throughput comparable to that of commercial tissue scanners while maintaining the nanoscale sensitivity to morphology. Based on the measured phase information, we implemented software tools for autofocusing during imaging, as well as image archiving and data access. To illustrate the potential of our technology for large volume pathology screening, we established an "intrinsic marker" for colorectal disease that detects tissue with dysplasia or colorectal cancer and flags specific areas for further examination, potentially improving the efficiency of existing pathology workflows.

Escherichia coli cell-free protein synthesis and isotope labeling of mammalian proteins.

Science.gov (United States)

Terada, Takaho; Yokoyama, Shigeyuki

2015-01-01

This chapter describes the cell-free protein synthesis method, using an Escherichia coli cell extract. This is a cost-effective method for milligram-scale protein production and is particularly useful for the production of mammalian proteins, protein complexes, and membrane proteins that are difficult to synthesize by recombinant expression methods, using E. coli and eukaryotic cells. By adjusting the conditions of the cell-free method, zinc-binding proteins, disulfide-bonded proteins, ligand-bound proteins, etc., may also be produced. Stable isotope labeling of proteins can be accomplished by the cell-free method, simply by using stable isotope-labeled amino acid(s) in the cell-free reaction. Moreover, the cell-free protein synthesis method facilitates the avoidance of stable isotope scrambling and dilution over the recombinant expression methods and is therefore advantageous for amino acid-selective stable isotope labeling. Site-specific stable isotope labeling is also possible with a tRNA molecule specific to the UAG codon. By the cell-free protein synthesis method, coupled transcription-translation is performed from a plasmid vector or a PCR-amplified DNA fragment encoding the protein. A milligram quantity of protein can be produced with a milliliter-scale reaction solution in the dialysis mode. More than a thousand solution structures have been determined by NMR spectroscopy for uniformly labeled samples of human and mouse functional domain proteins, produced by the cell-free method. Here, we describe the practical aspects of mammalian protein production by the cell-free method for NMR spectroscopy. © 2015 Elsevier Inc. All rights reserved.

Cerebrospinal Fluid Proteome of Patients with Acute Lyme Disease

Energy Technology Data Exchange (ETDEWEB)

Angel, Thomas E.; Jacobs, Jon M.; Smith, Robert P.; Pasternack, Mark S.; Elias, Susan; Gritsenko, Marina A.; Shukla, Anil K.; Gilmore, Edward C.; McCarthy, Carol; Camp, David G.; Smith, Richard D.

2012-10-05

Acute Lyme disease results from transmission of and infection by the bacterium Borrelia burgdorferi following a tick bite. During acute infection, bacteria can disseminate to the central nervous system (CNS) leading to the development of Lyme meningitis. Here we have analyzed pooled cerebrospinal fluid (CSF) allowing for a deep view into the proteome for a cohort of patients with early-disseminated Lyme disease and CSF inflammation leading to the identification of proteins that reflect host responses, which are distinct for subjects with acute Lyme disease. Additionally, we analyzed individual patient samples and quantified changes in protein abundance employing label-free quantitative mass spectrometry based methods. The measured changes in protein abundances reflect the impact of acute Lyme disease on the CNS as presented in CSF. We have identified 89 proteins that differ significantly in abundance in patients with acute Lyme disease. A number of the differentially abundant proteins have been found to be localized to brain synapse and thus constitute important leads for better understanding of the neurological consequence of disseminated Lyme disease.

Quantitative Proteomics for the Comprehensive Analysis of Stress Responses of Lactobacillus paracasei subsp. paracasei F19.

Science.gov (United States)

Schott, Ann-Sophie; Behr, Jürgen; Geißler, Andreas J; Kuster, Bernhard; Hahne, Hannes; Vogel, Rudi F

2017-10-06

Lactic acid bacteria are broadly employed as starter cultures in the manufacture of foods. Upon technological preparation, they are confronted with drying stress that amalgamates numerous stress conditions resulting in losses of fitness and survival. To better understand and differentiate physiological stress responses, discover general and specific markers for the investigated stress conditions, and predict optimal preconditioning for starter cultures, we performed a comprehensive genomic and quantitative proteomic analysis of a commonly used model system, Lactobacillus paracasei subsp. paracasei TMW 1.1434 (isogenic with F19) under 11 typical stress conditions, including among others oxidative, osmotic, pH, and pressure stress. We identified and quantified >1900 proteins in triplicate analyses, representing 65% of all genes encoded in the genome. The identified genes were thoroughly annotated in terms of subcellular localization prediction and biological functions, suggesting unbiased and comprehensive proteome coverage. In total, 427 proteins were significantly differentially expressed in at least one condition. Most notably, our analysis suggests that optimal preconditioning toward drying was predicted to be alkaline and high-pressure stress preconditioning. Taken together, we believe the presented strategy may serve as a prototypic example for the analysis and utility of employing quantitative-mass-spectrometry-based proteomics to study bacterial physiology.

Integrative Analysis of Subcellular Quantitative Proteomics Studies Reveals Functional Cytoskeleton Membrane-Lipid Raft Interactions in Cancer.

Science.gov (United States)

Shah, Anup D; Inder, Kerry L; Shah, Alok K; Cristino, Alexandre S; McKie, Arthur B; Gabra, Hani; Davis, Melissa J; Hill, Michelle M

2016-10-07

Lipid rafts are dynamic membrane microdomains that orchestrate molecular interactions and are implicated in cancer development. To understand the functions of lipid rafts in cancer, we performed an integrated analysis of quantitative lipid raft proteomics data sets modeling progression in breast cancer, melanoma, and renal cell carcinoma. This analysis revealed that cancer development is associated with increased membrane raft-cytoskeleton interactions, with ∼40% of elevated lipid raft proteins being cytoskeletal components. Previous studies suggest a potential functional role for the raft-cytoskeleton in the action of the putative tumor suppressors PTRF/Cavin-1 and Merlin. To extend the observation, we examined lipid raft proteome modulation by an unrelated tumor suppressor opioid binding protein cell-adhesion molecule (OPCML) in ovarian cancer SKOV3 cells. In agreement with the other model systems, quantitative proteomics revealed that 39% of OPCML-depleted lipid raft proteins are cytoskeletal components, with microfilaments and intermediate filaments specifically down-regulated. Furthermore, protein-protein interaction network and simulation analysis showed significantly higher interactions among cancer raft proteins compared with general human raft proteins. Collectively, these results suggest increased cytoskeleton-mediated stabilization of lipid raft domains with greater molecular interactions as a common, functional, and reversible feature of cancer cells.

Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

DEFF Research Database (Denmark)

Cox, T.R.; Schoof, Erwin; Gartland, A.

2015-01-01

secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic...... and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397...

Labeling of indocyanine green with carrier-free iodine-123

International Nuclear Information System (INIS)

Ansari, A.N.; Lambrecht, R.M.; Redvanly, C.S.; Wolf, A.P.

1976-01-01

The method is described for labeling indocyanine green (ICG) with carrier-free iodine-123 by condensing xenon-123 on crystals of ICG followed by permitting decay of the 123 Xe a sufficient length of time to produce 123 I-electronically excited ions and atoms which subsequently label ICG. 4 claims, no drawings

Comparative proteome approach demonstrates that platelet-derived growth factor C and D efficiently induce proliferation while maintaining multipotency of hMSCs

Energy Technology Data Exchange (ETDEWEB)

Sotoca, Ana M., E-mail: a.sotoca@science.ru.nl [Department of Cell and Applied Biology, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Roelofs-Hendriks, Jose [Department of Cell and Applied Biology, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen (Netherlands); Boeren, Sjef [Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen (Netherlands); Kraan, Peter M. van der [Department of Rheumatology Research and Advanced Therapeutics, Radboud University Nijmegen Medical Centre, Nijmegen (Netherlands); Vervoort, Jacques [Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA Wageningen (Netherlands); Zoelen, Everardus J.J. van; Piek, Ester [Department of Cell and Applied Biology, Radboud University, Heijendaalseweg 135, 6525 AJ Nijmegen (Netherlands)

2013-10-15

This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function. - Highlights: • PDGFs (C and D) significantly increased the number of multipotent undifferentiated hMSCs. • Enhanced proliferation did not impair the ability to undergo lineage-specific differentiation. • Proteomic analysis confirmed the overall signatures of the ‘intact’ cells.

Proteomic Analysis of the Endosperm Ontogeny of Jatropha curcas L. Seeds.

Science.gov (United States)

Shah, Mohibullah; Soares, Emanoella L; Carvalho, Paulo C; Soares, Arlete A; Domont, Gilberto B; Nogueira, Fábio C S; Campos, Francisco A P

2015-06-05

Seeds of Jatropha curcas L. represent a potential source of raw material for the production of biodiesel. However, this use is hampered by the lack of basic information on the biosynthetic pathways associated with synthesis of toxic diterpenes, fatty acids, and triacylglycerols, as well as the pattern of deposition of storage proteins during seed development. In this study, we performed an in-depth proteome analysis of the endosperm isolated from five developmental stages which resulted in the identification of 1517, 1256, 1033, 752, and 307 proteins, respectively, summing up 1760 different proteins. Proteins with similar label free quantitation expression pattern were grouped into five clusters. The biological significance of these identifications is discussed with special focus on the analysis of seed storage proteins, proteins involved in the metabolism of fatty acids, carbohydrates, toxic components and proteolytic processing. Although several enzymes belonging to the biosynthesis of diterpenoid precursors were identified, we were unable to find any terpene synthase/cyclase, indicating that the synthesis of phorbol esters, the main toxic diterpenes, does not occur in seeds. The strategy used enabled us to provide a first in depth proteome analysis of the developing endosperm of this biodiesel plant, providing an important glimpse into the enzymatic machinery devoted to the production of C and N sources to sustain seed development.

Label-free monitoring of diffusion in microfluidics

DEFF Research Database (Denmark)

Sørensen, Kristian Tølbøl; Kristensen, Anders

2017-01-01

Label-free, real-time detection of concentration gradients is demonstrated in a microfluidic H-filter, using an integrated photonic crystal slab sensor to monitor sample refractive index with spatial resolution. The recorded diffusion profiles reveal root-mean-square diffusion lengths for non...

Label and label-free based surface-enhanced Raman scattering for pathogen bacteria detection: A review.

Science.gov (United States)

Liu, Yu; Zhou, Haibo; Hu, Ziwei; Yu, Guangxia; Yang, Danting; Zhao, Jinshun

2017-08-15

Rapid, accurate detection of pathogen bacteria is a highly topical research area for the sake of food safety and public health. Surface-enhanced Raman scattering (SERS) is being considered as a powerful and attractive technique for pathogen bacteria detection, due to its sensitivity, high speed, comparatively low cost, multiplexing ability and portability. This contribution aims to give a comprehensive overview of SERS as a technique for rapid detection of pathogen bacteria based on label and label-free strategies. A brief tutorial on SERS is given first of all. Then we summarize the recent trends and developments of label and label-free based SERS applied to detection of pathogen bacteria, including the relatively complete interpretation of SERS spectra. In addition, multifunctional SERS platforms for pathogen bacteria in matrix are discussed as well. Furthermore, an outlook of the work done and a perspective on the future directions of SERS as a reliable tool for real-time pathogen bacteria detection are given. Copyright © 2017 Elsevier B.V. All rights reserved.

Clinical proteomics: Current status, challenges, and future perspectives

Directory of Open Access Journals (Sweden)

Shyh-Horng Chiou

2011-01-01

Full Text Available This account will give an overview and evaluation of the current advances in mass spectrometry (MS-based proteomics platforms and technology. A general review of some background information concerning the application of these methods in the characterization of molecular sizes and related protein expression profiles associated with different types of cells under varied experimental conditions will be presented. It is intended to provide a concise and succinct overview to those clinical researchers first exposed to this foremost powerful methodology in modern life sciences of postgenomic era. Proteomic characterization using highly sophisticated and expensive instrumentation of MS has been used to characterize biological samples of complex protein mixtures with vastly different protein structure and composition. These systems are then used to highlight the versatility and potential of the MS-based proteomic strategies for facilitating protein expression analysis of various disease-related organisms or tissues of interest. Major MS-based strategies reviewed herein include (1 matrix-assisted laser desorption ionization-MS and electron-spray ionization proteomics; (2 one-dimensional or two-dimensional gel-based proteomics; (3 gel-free shotgun proteomics in conjunction with liquid chromatography/tandem MS; (4 Multiple reaction monitoring coupled tandem MS quantitative proteomics and; (5 Phosphoproteomics based on immobilized metal affinity chromatography and liquid chromatography-MS/MS.

HoloMonitor M4: holographic imaging cytometer for real-time kinetic label-free live-cell analysis of adherent cells

Science.gov (United States)

Sebesta, Mikael; Egelberg, Peter J.; Langberg, Anders; Lindskov, Jens-Henrik; Alm, Kersti; Janicke, Birgit

2016-03-01

Live-cell imaging enables studying dynamic cellular processes that cannot be visualized in fixed-cell assays. An increasing number of scientists in academia and the pharmaceutical industry are choosing live-cell analysis over or in addition to traditional fixed-cell assays. We have developed a time-lapse label-free imaging cytometer HoloMonitorM4. HoloMonitor M4 assists researchers to overcome inherent disadvantages of fluorescent analysis, specifically effects of chemical labels or genetic modifications which can alter cellular behavior. Additionally, label-free analysis is simple and eliminates the costs associated with staining procedures. The underlying technology principle is based on digital off-axis holography. While multiple alternatives exist for this type of analysis, we prioritized our developments to achieve the following: a) All-inclusive system - hardware and sophisticated cytometric analysis software; b) Ease of use enabling utilization of instrumentation by expert- and entrylevel researchers alike; c) Validated quantitative assay end-points tracked over time such as optical path length shift, optical volume and multiple derived imaging parameters; d) Reliable digital autofocus; e) Robust long-term operation in the incubator environment; f) High throughput and walk-away capability; and finally g) Data management suitable for single- and multi-user networks. We provide examples of HoloMonitor applications of label-free cell viability measurements and monitoring of cell cycle phase distribution.

iTRAQ-Based Quantitative Proteomic Analysis of the Initiation of Head Regeneration in Planarians.

Directory of Open Access Journals (Sweden)

Xiaofang Geng

Full Text Available The planarian Dugesia japonica has amazing ability to regenerate a head from the anterior ends of the amputated stump with maintenance of the original anterior-posterior polarity. Although planarians present an attractive system for molecular investigation of regeneration and research has focused on clarifying the molecular mechanism of regeneration initiation in planarians at transcriptional level, but the initiation mechanism of planarian head regeneration (PHR remains unclear at the protein level. Here, a global analysis of proteome dynamics during the early stage of PHR was performed using isobaric tags for relative and absolute quantitation (iTRAQ-based quantitative proteomics strategy, and our data are available via ProteomeXchange with identifier PXD002100. The results showed that 162 proteins were differentially expressed at 2 h and 6 h following amputation. Furthermore, the analysis of expression patterns and functional enrichment of the differentially expressed proteins showed that proteins involved in muscle contraction, oxidation reduction and protein synthesis were up-regulated in the initiation of PHR. Moreover, ingenuity pathway analysis showed that predominant signaling pathways such as ILK, calcium, EIF2 and mTOR signaling which were associated with cell migration, cell proliferation and protein synthesis were likely to be involved in the initiation of PHR. The results for the first time demonstrated that muscle contraction and ILK signaling might played important roles in the initiation of PHR at the global protein level. The findings of this research provide a molecular basis for further unraveling the mechanism of head regeneration initiation in planarians.

Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway

DEFF Research Database (Denmark)

Gruhler, Albrecht; Olsen, Jesper Velgaard; Mohammed, Shabaz

2005-01-01

of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS....... Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified...

Quantitative Tissue Proteomics Analysis Reveals Versican as Potential Biomarker for Early-Stage Hepatocellular Carcinoma.

Science.gov (United States)

Naboulsi, Wael; Megger, Dominik A; Bracht, Thilo; Kohl, Michael; Turewicz, Michael; Eisenacher, Martin; Voss, Don Marvin; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

2016-01-04

Hepatocellular carcinoma (HCC) is one of the most aggressive tumors, and the treatment outcome of this disease is improved when the cancer is diagnosed at an early stage. This requires biomarkers allowing an accurate and early tumor diagnosis. To identify potential markers for such applications, we analyzed a patient cohort consisting of 50 patients (50 HCC and 50 adjacent nontumorous tissue samples as controls) using two independent proteomics approaches. We performed label-free discovery analysis on 19 HCC and corresponding tissue samples. The data were analyzed considering events known to take place in early events of HCC development, such as abnormal regulation of Wnt/b-catenin and activation of receptor tyrosine kinases (RTKs). 31 proteins were selected for verification experiments. For this analysis, the second set of the patient cohort (31 HCC and corresponding tissue samples) was analyzed using selected (multiple) reaction monitoring (SRM/MRM). We present the overexpression of ATP-dependent RNA helicase (DDX39), Fibulin-5 (FBLN5), myristoylated alanine-rich C-kinase substrate (MARCKS), and Serpin H1 (SERPINH1) in HCC for the first time. We demonstrate Versican core protein (VCAN) to be significantly associated with well differentiated and low-stage HCC. We revealed for the first time the evidence of VCAN as a potential biomarker for early-HCC diagnosis.

Label-free detection of insulin and glucagon within human islets of Langerhans using Raman spectroscopy.

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Janneke Hilderink

Full Text Available Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm(-1 band assigned to disulfide bridges in insulin, and the 1552 cm(-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1 of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans.

Free-solution, label-free molecular interactions studied by back-scattering interferometry

DEFF Research Database (Denmark)

Bornhop, D.J.; Latham, J.C.; Kussrow, A.

2007-01-01

Free-solution, label-free molecular interactions were investigated with back-scattering interferometry in a simple optical train composed of a helium-neon laser, a microfluidic channel, and a position sensor. Molecular binding interactions between proteins, ions and protein, and small molecules...... and protein, were determined with high dynamic range dissociation constants (K-d spanning six decades) and unmatched sensitivity (picomolar K-d's and detection limits of 10,000s of molecules). With this technique, equilibrium dissociation constants were quantified for protein A and immunoglobulin G...

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

Energy Technology Data Exchange (ETDEWEB)

Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei, E-mail: biehzw@nus.edu.sg [Optical Bioimaging Laboratory, Department of Biomedical Engineering, Faculty of Engineering, National University of Singapore, Singapore 117576 (Singapore)

2014-09-08

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Label-free three-dimensional imaging of cell nucleus using third-harmonic generation microscopy

International Nuclear Information System (INIS)

Lin, Jian; Zheng, Wei; Wang, Zi; Huang, Zhiwei

2014-01-01

We report the implementation of the combined third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) microscopy for label-free three-dimensional (3-D) imaging of cell nucleus morphological changes in liver tissue. THG imaging shows regular spherical shapes of normal hepatocytes nuclei with inner chromatin structures while revealing the condensation of chromatins and nuclear fragmentations in hepatocytes of diseased liver tissue. Colocalized THG and TPEF imaging provides complementary information of cell nuclei and cytoplasm in tissue. This work suggests that 3-D THG microscopy has the potential for quantitative analysis of nuclear morphology in cells at a submicron-resolution without the need for DNA staining.

Clinical proteomics

DEFF Research Database (Denmark)

Albrethsen, Jakob; Frederiksen, Hanne; Johannsen, Trine Holm

2018-01-01

Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS)-platforms...... standards and calibrants. The present challenge is to examine if targeted proteomics of IGF-I can truly measure up to the routine performance that must be expected from a clinical testing platform.......Clinical proteomics aims to deliver cost-effective multiplexing of potentially hundreds of diagnostic proteins, including distinct protein isoforms. The analytical strategy known as targeted proteomics is particularly promising because it is compatible with robust mass spectrometry (MS......)-platforms already implemented in many clinical laboratories for routine quantitation of small molecules (i.e. uHPLC coupled to triple-quadrupole MS). Progress in targeted proteomics of circulating insulin-like growth factor 1 (IGF-I) have provided valuable insights about tryptic peptides, transitions, internal...

Quantitative proteomics by amino acid labeling identifies novel NHR-49 regulated proteins in C. elegans

DEFF Research Database (Denmark)

Fredens, Julius; Færgeman, Nils J.

2012-01-01

in the nematode Caenorhabditis elegans. We have recently shown that C. elegans can be completely labeled with heavy-labeled lysine by feeding worms on prelabeled lysine auxotroph Escherichia coli for just one generation. We applied this methodology to examine the organismal response to functional loss or RNAi...... gene knockdown by RNAi provides a powerful tool with broad implications for C. elegans biology....

Identification of cypermethrin induced protein changes in green algae by iTRAQ quantitative proteomics.

Science.gov (United States)

Gao, Yan; Lim, Teck Kwang; Lin, Qingsong; Li, Sam Fong Yau

2016-04-29

Cypermethrin (CYP) is one of the most widely used pesticides in large scale for agricultural and domestic purpose and the residue often seriously affects aquatic system. Environmental pollutant-induced protein changes in organisms could be detected by proteomics, leading to discovery of potential biomarkers and understanding of mode of action. While proteomics investigations of CYP stress in some animal models have been well studied, few reports about the effects of exposure to CYP on algae proteome were published. To determine CYP effect in algae, the impact of various dosages (0.001μg/L, 0.01μg/L and 1μg/L) of CYP on green algae Chlorella vulgaris for 24h and 96h was investigated by using iTRAQ quantitative proteomics technique. A total of 162 and 198 proteins were significantly altered after CYP exposure for 24h and 96h, respectively. Overview of iTRAQ results indicated that the influence of CYP on algae protein might be dosage-dependent. Functional analysis of differentially expressed proteins showed that CYP could induce protein alterations related to photosynthesis, stress responses and carbohydrate metabolism. This study provides a comprehensive view of complex mode of action of algae under CYP stress and highlights several potential biomarkers for further investigation of pesticide-exposed plant and algae. Copyright © 2016 Elsevier B.V. All rights reserved.

Investigating the Effect of Growth Phase on the Surface-Layer Associated Proteome of Lactobacillus acidophilus Using Quantitative Proteomics

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Courtney Klotz

2017-11-01

Full Text Available Bacterial surface-layers (S-layers are semi-porous crystalline arrays that self-assemble to form the outermost layer of some cell envelopes. S-layers have been shown to act as scaffolding structures for the display of auxiliary proteins externally. These S-layer associated proteins have recently gained attention in probiotics due to their direct physical contact with the intestinal mucosa and potential role in cell proliferation, adhesion, and immunomodulation. A number of studies have attempted to catalog the S-layer associated proteome of Lactobacillus acidophilus NCFM under a single condition. However, due to the versatility of the cell surface, we chose to employ a multiplexing-based approach with the intention of accurately contrasting multiple conditions. In this study, a previously described lithium chloride isolation protocol was used to release proteins bound to the L. acidophilus S-layer during logarithmic and early stationary growth phases. Protein quantification values were obtained via TMT (tandem mass tag labeling combined with a triple-stage mass spectrometry (MS3 method. Results showed significant growth stage-dependent alterations to the surface-associated proteome while simultaneously highlighting the sensitivity and reproducibility of the technology. Thus, this study establishes a framework for quantifying condition-dependent changes to cell surface proteins that can easily be applied to other S-layer forming bacteria.

Quantitative proteomics of Chlorobaculum tepidum

DEFF Research Database (Denmark)

Falkenby, Lasse Gaarde; Szymanska, Monika; Holkenbrink, Carina

2011-01-01

Chlorobaculum (Cba.) tepidum is a green sulfur bacterium that oxidizes sulfide, elemental sulfur, and thiosulfate for photosynthetic growth. To gain insight into the sulfur metabolism, the proteome of Cba. tepidum cells sampled under different growth conditions has been quantified using a rapid g...

Quantitative analysis of oyster larval proteome provides new insights into the effects of multiple climate change stressors

KAUST Repository

Dineshram, Ramadoss; Chandramouli, Kondethimmanahalli; Ko, Ginger Wai Kuen; Zhang, Huoming; Qian, Pei-Yuan; Ravasi, Timothy; Thiyagarajan, Vengatesen

2016-01-01

might be affected in a future ocean, we examined changes in the proteome of metamorphosing larvae under multiple stressors: decreased pH (pH 7.4), increased temperature (30 °C), and reduced salinity (15 psu). Quantitative protein expression profiling

Quantitation of images from a multiwire camera for autoradiography of tritium-labelled substances

International Nuclear Information System (INIS)

Lockett, S.J.; Ramsden, D.B.; Bradwell, A.R.

1987-01-01

It has been shown that tritium-labelled substances in two-dimensional systems can be quantitated using a multiwire camera. Its accuracy has now been improved by correcting results for non-uniformity of response over the detection area. Uniformity was assessed by imaging plates of nominally uniform activity. The results were then used to correct images from plates containing tritium-labelled proteins using a computer program. Errors were reduced from 11.3 (+ -6.1) to 7.7 (+ - 2.8)% for standard sources and from 6.2 (+ - 1.8) to 1.9 (+ -0.6)% for a plate containing the labelled proteins. The conducting carbon layer covering the plate absorbed 36 (+ - 3)% of the tritium beta radiation and was estimated to be 85 nm in thickness. Quantitation of the labelled proteins by the camera gave a good correlation with protein content (chi-squared: 30-40%). The activities of the protein samples were measured to an accuracy of 10% by comparison with standard sources. These results indicate useful quantitation of tritiated compounds in two-dimensional media using the multiwire camera. (author)

Proteomic Analysis of Lipid Raft-Like Detergent-Resistant Membranes of Lens Fiber Cells.

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Wang, Zhen; Schey, Kevin L

2015-12-01

Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome. Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis. A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains. These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.

Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

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Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

2012-12-01

What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To

Annotation of loci from genome-wide association studies using tissue-specific quantitative interaction proteomics

DEFF Research Database (Denmark)

Lundby, Alicia; Rossin, Elizabeth J.; Steffensen, Annette B.

2014-01-01

Genome-wide association studies (GWAS) have identified thousands of loci associated with complex traits, but it is challenging to pinpoint causal genes in these loci and to exploit subtle association signals. We used tissue-specific quantitative interaction proteomics to map a network of five genes...... involved in the Mendelian disorder long QT syndrome (LOTS). We integrated the LOTS network with GWAS loci from the corresponding common complex trait, QT-interval variation, to identify candidate genes that were subsequently confirmed in Xenopus laevis oocytes and zebrafish. We used the LOTS protein...... network to filter weak GWAS signals by identifying single-nucleotide polymorphisms (SNPs) in proximity to genes in the network supported by strong proteomic evidence. Three SNPs passing this filter reached genome-wide significance after replication genotyping. Overall, we present a general strategy...

Pollen developmental defects in ZD-CMS rice line explored by cytological, molecular and proteomic approaches.

Science.gov (United States)

Yan, Junjie; Tian, Han; Wang, Shuzhen; Shao, Jinzhen; Zheng, Yinzhen; Zhang, Hongyuan; Guo, Lin; Ding, Yi

2014-08-28

Cytoplasmic male sterility (CMS) is a widely observed phenomenon, which is especially useful in hybrid seed production. Meixiang A (MxA) is a new rice CMS line derived from a pollen-free sterile line named Yunnan ZidaoA (ZD-CMS). In this study, a homologous WA352 gene with variation in two nucleotides was identified in MxA. Cytological analysis revealed that MxA was aborted in the early uninucleate stage. The protein expression profiles of MxA and its maintainer line MeixiangB (MxB) were systematically compared using iTRAQ-based quantitative proteomics technology using young florets at the early uninucleate stage. A total of 688 proteins were quantified in both rice lines, and 45 of these proteins were found to be differentially expressed. Bioinformatics analysis indicated a large number of the proteins involved in carbohydrate metabolism or the stress response were downregulated in MxA, suggesting that these metabolic processes had been hindered during pollen development in MxA. The ROS (reactive oxygen species) level was increased in the mitochondrion of MxA, and further ultrastructural analysis showed the mitochondria with disrupted cristae in the rice CMS line MxA. These findings substantially contribute to our knowledge of pollen developmental defects in ZD-CMS rice line. MeixiangA (MxA) is a new type of rice CMS line, which is derived from pollen-free sterile line Yunnan ZidaoA. In this study, the cytological, molecular and proteomic approaches were used to study the characteristics of this new CMS line. Cytological study indicates the CMS line is aborted at the early uninucleate stage. A potential sterile gene ZD352 is identified in MxA, the protein product of which is mainly accumulated at the MMC/Meiotic stage. iTRAQ based proteomic analysis is performed to study the relevant proteins involved in the CMS occurance, 45 proteins are found to be significant differentially expressed and these proteins are involved in many cellular processes such as

Proteomics of apheresis platelet supernatants during routine storage: Gender-related differences.

Science.gov (United States)

Dzieciatkowska, Monika; D'Alessandro, Angelo; Burke, Timothy A; Kelher, Marguerite R; Moore, Ernest E; Banerjee, Anirban; Silliman, Christopher C; West, Bernadette F; Hansen, Kirk C

2015-01-01

Proteomics has identified potential pathways involved in platelet storage lesions, which correlate with untoward effects in the recipient, including febrile non-haemolytic reactions. We hypothesize that an additional pathway involves protein mediators that accumulate in the platelet supernatants during routine storage in a donor gender-specific fashion. Apheresis platelet concentrates were collected from 5 healthy males and 5 females and routinely stored. The 14 most abundant plasma proteins were removed and the supernatant proteins from days 1 and 5 were analyzed via 1D-SDS-PAGE/nanoLC-MS/MS, before label-free quantitative proteomics analyses. Findings from a subset of 18 proteins were validated via LC-SRM analyses against stable isotope labeled standards. A total of 503 distinct proteins were detected in the platelet supernatants from the 4 sample groups: female or male donor platelets, either at storage day 1 or 5. Proteomics suggested a storage and gender-dependent impairment of blood coagulation mediators, pro-inflammatory complement components and cytokines, energy and redox metabolic enzymes. The supernatants from female donors demonstrated increased deregulation of structural proteins, extracellular matrix proteins and focal adhesion proteins, possibly indicating storage-dependent platelet activation. Routine storage of platelet concentrates induces changes in the supernatant proteome, which may have effects on the transfused patient, some of which are related to donor gender. The rationale behind this study is that protein components in platelet releasates have been increasingly observed to play a key role in adverse events and impaired homeostasis in transfused recipients. In this view, proteomics has recently emerged as a functional tool to address the issue of protein composition of platelet releasates from buffy coat-derived platelet concentrates in the blood bank. Despite early encouraging studies on buffy coat-derived platelet concentrates, platelet

Research resource: new and diverse substrates for the insulin receptor isoform a revealed by quantitative proteomics after stimulation with igf-ii or insulin

DEFF Research Database (Denmark)

Morcavallo, Alaide; Gaspari, Marco; Pandini, Giuseppe

2011-01-01

progression. We hypothesized that IGF-II binding to the IR-A elicits a unique signaling pathway. In order to obtain an unbiased evaluation of IR-A substrates differentially involved after IGF-II and insulin stimulation, we performed quantitative proteomics of IR-A substrates recruited to tyrosine......-phosphorylated protein complexes using stable isotope labeling with amino acids in cell culture in combination with antiphosphotyrosine antibody pull down and mass spectrometry. Using cells expressing only the human IR-A and lacking the IGF-I receptor, we identified 38 IR-A substrates. Only 10 were known IR mediators......, whereas 28 substrates were not previously related to IR signaling. Eleven substrates were recruited by stimulation with both ligands: two equally recruited by IGF-II and insulin, three more strongly recruited by IGF-II, and six more strongly recruited by insulin. Moreover, 14 substrates were recruited...

Mass spectrometry-based proteomics: basic principles and emerging technologies and directions.

Science.gov (United States)

Van Riper, Susan K; de Jong, Ebbing P; Carlis, John V; Griffin, Timothy J

2013-01-01

As the main catalytic and structural molecules within living systems, proteins are the most likely biomolecules to be affected by radiation exposure. Proteomics, the comprehensive characterization of proteins within complex biological samples, is therefore a research approach ideally suited to assess the effects of radiation exposure on cells and tissues. For comprehensive characterization of proteomes, an analytical platform capable of quantifying protein abundance, identifying post-translation modifications and revealing members of protein complexes on a system-wide level is necessary. Mass spectrometry (MS), coupled with technologies for sample fractionation and automated data analysis, provides such a versatile and powerful platform. In this chapter we offer a view on the current state of MS-proteomics, and focus on emerging technologies within three areas: (1) New instrumental methods; (2) New computational methods for peptide identification; and (3) Label-free quantification. These emerging technologies should be valuable for researchers seeking to better understand biological effects of radiation on living systems.

Prospects and challenges of quantitative phase imaging in tumor cell biology

Science.gov (United States)

Kemper, Björn; Götte, Martin; Greve, Burkhard; Ketelhut, Steffi

2016-03-01

Quantitative phase imaging (QPI) techniques provide high resolution label-free quantitative live cell imaging. Here, prospects and challenges of QPI in tumor cell biology are presented, using the example of digital holographic microscopy (DHM). It is shown that the evaluation of quantitative DHM phase images allows the retrieval of different parameter sets for quantification of cellular motion changes in migration and motility assays that are caused by genetic modifications. Furthermore, we demonstrate simultaneously label-free imaging of cell growth and morphology properties.

Integrative proteomics and tissue microarray profiling indicate the association between overexpressed serum proteins and non-small cell lung cancer.

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Yansheng Liu

Full Text Available Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA and Multiple reaction monitoring (MRM assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG and Leucine-rich alpha-2-glycoprotein (LRG1, two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.

Concurrent Label-Free Mass Spectrometric Analysis of Dystrophin Isoform Dp427 and the Myofibrosis Marker Collagen in Crude Extracts from mdx-4cv Skeletal Muscles

Science.gov (United States)

Murphy, Sandra; Zweyer, Margit; Mundegar, Rustam R.; Henry, Michael; Meleady, Paula; Swandulla, Dieter; Ohlendieck, Kay

2015-01-01

The full-length dystrophin protein isoform of 427 kDa (Dp427), the absence of which represents the principal abnormality in X-linked muscular dystrophy, is difficult to identify and characterize by routine proteomic screening approaches of crude tissue extracts. This is probably related to its large molecular size, its close association with the sarcolemmal membrane, and its existence within a heterogeneous glycoprotein complex. Here, we used a careful extraction procedure to isolate the total protein repertoire from normal versus dystrophic mdx-4cv skeletal muscles, in conjunction with label-free mass spectrometry, and successfully identified Dp427 by proteomic means. In contrast to a considerable number of previous comparative studies of the total skeletal muscle proteome, using whole tissue proteomics we show here for the first time that the reduced expression of this membrane cytoskeletal protein is the most significant alteration in dystrophinopathy. This agrees with the pathobiochemical concept that the almost complete absence of dystrophin is the main defect in Duchenne muscular dystrophy and that the mdx-4cv mouse model of dystrophinopathy exhibits only very few revertant fibers. Significant increases in collagens and associated fibrotic marker proteins, such as fibronectin, biglycan, asporin, decorin, prolargin, mimecan, and lumican were identified in dystrophin-deficient muscles. The up-regulation of collagen in mdx-4cv muscles was confirmed by immunofluorescence microscopy and immunoblotting. Thus, this is the first mass spectrometric study of crude tissue extracts that puts the proteomic identification of dystrophin in its proper pathophysiological context. PMID:28248273

Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

KAUST Repository

Zhang, Huoming

2010-07-02

Human monocytes\\' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

Study of monocyte membrane proteome perturbation during lipopolysaccharide-induced tolerance using iTRAQ-based quantitative proteomic approach

KAUST Repository

Zhang, Huoming; Zhao, Changqing; Li, Xin; Zhu, Yi; Gan, Chee Sian; Wang, Yong; Ravasi, Timothy; Qian, Pei-Yuan; Wong, Siew Cheng; Sze, Siu Kwan

2010-01-01

Human monocytes' exposure to low-level lipopolysaccharide (LPS) induces temporary monocytic insensitivity to subsequent LPS challenge. The underlying mechanism of this phenomenon could have important clinical utilities in preventing and/or treating severe infections. In this study, we used an iTRAQ-based quantitative proteomic approach to comprehensively characterize the membrane proteomes of monocytes before and after LPS exposure. We identified a total of 1651 proteins, of which 53.6% were membrane proteins. Ninety-four percent of the proteins were quantified and 255 proteins were shown to be tightly regulated by LPS. Subcellular location analysis revealed organelle-specific response to LPS exposure: more than 90% of identified mitochondrial membrane proteins were significant downregulated, whereas the majority of proteins from other organelles such as ER, Golgi and ribosome were upregulated. Moreover, we found that the expression of most receptors potentially involved in LPS signal pathway (CD14, toll-like receptor 4, CD11/CD18 complex) were substantially decreased, while the expression of molecules involved in LPS neutralization were enhanced after LPS challenge. Together, these findings could be of significance in understanding the mechanism of LPS tolerance and provide values for designing new approaches for regulating monocytic responses in sepsis patients.

Label-Free Electrical Detection Using Carbon Nanotube-Based Biosensors

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Kenzo Maehashi

2009-07-01

Full Text Available Label-free detections of biomolecules have attracted great attention in a lot of life science fields such as genomics, clinical diagnosis and practical pharmacy. In this article, we reviewed amperometric and potentiometric biosensors based on carbon nanotubes (CNTs. In amperometric detections, CNT-modified electrodes were used as working electrodes to significantly enhance electroactive surface area. In contrast, the potentiometric biosensors were based on aptamer-modified CNT field-effect transistors (CNTFETs. Since aptamers are artificial oligonucleotides and thus are smaller than the Debye length, proteins can be detected with high sensitivity. In this review, we discussed on the technology, characteristics and developments for commercialization in label-free CNT-based biosensors.

Systematic and quantitative comparison of digest efficiency and specificity reveals the impact of trypsin quality on MS-based proteomics.

Science.gov (United States)

Burkhart, Julia Maria; Schumbrutzki, Cornelia; Wortelkamp, Stefanie; Sickmann, Albert; Zahedi, René Peiman

2012-02-02

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.

Drought-Induced Leaf Proteome Changes in Switchgrass Seedlings

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Zhujia Ye

2016-08-01

Full Text Available Switchgrass (Panicum virgatum is a perennial crop producing deep roots and thus highly tolerant to soil water deficit conditions. However, seedling establishment in the field is very susceptible to prolonged and periodic drought stress. In this study, a “sandwich” system simulating a gradual water deletion process was developed. Switchgrass seedlings were subjected to a 20-day gradual drought treatment process when soil water tension was increased to 0.05 MPa (moderate drought stress and leaf physiological properties had expressed significant alteration. Drought-induced changes in leaf proteomes were identified using the isobaric tags for relative and absolute quantitation (iTRAQ labeling method followed by nano-scale liquid chromatography mass spectrometry (nano-LC-MS/MS analysis. Additionally, total leaf proteins were processed using a combinatorial library of peptide ligands to enrich for lower abundance proteins. Both total proteins and those enriched samples were analyzed to increase the coverage of the quantitative proteomics analysis. A total of 7006 leaf proteins were identified, and 257 (4% of the leaf proteome expressed a significant difference (p < 0.05, fold change <0.6 or >1.7 from the non-treated control to drought-treated conditions. These proteins are involved in the regulation of transcription and translation, cell division, cell wall modification, phyto-hormone metabolism and signaling transduction pathways, and metabolic pathways of carbohydrates, amino acids, and fatty acids. A scheme of abscisic acid (ABA-biosynthesis and ABA responsive signal transduction pathway was reconstructed using these drought-induced significant proteins, showing systemic regulation at protein level to deploy the respective mechanism. Results from this study, in addition to revealing molecular responses to drought stress, provide a large number of proteins (candidate genes that can be employed to improve switchgrass seedling growth and

Functionalized Polymer Microgel Particles Enable Customizable Production of Label-Free Sensor Arrays.

Science.gov (United States)

Lifson, Mark A; Carter, Jared A; Miller, Benjamin L

2015-08-04

Probe molecule immobilization onto surfaces is a critical step in the production of many analytical devices, including labeled and label-free microarrays. New methods to increase the density and uniformity of probe deposition have the potential to significantly enhance the ultimate limits of detection and reproducibility. Hydrogel-based materials have been employed in the past to provide a 3D protein-friendly surface for deposition of antibodies and nucleic acids. However, these methods are susceptible to variation during polymerization of the hydrogel scaffold and provide limited opportunities for tuning deposition parameters on an antibody-by-antibody basis. In this work, a versatile hydrogel nanoparticle deposition method was developed for the production of label-free microarrays and tested in the context of antibody-antigen binding. Poly(N-isopropylacrylamide) nanoparticles (PNIPAM) were conjugated to antibodies using an avidin/biotin system and deposited onto surfaces using a noncontact printing system. After drying, these gel spots formed uniform and thin layers <10 nm in height. The conjugates were characterized with dynamic light scattering, scanning electron microscopy, and atomic force microscopy. We tested this format in the context of tumor necrosis factor-alpha (TNF-α) detection via arrayed imaging reflectometry (AIR), a label-free protein microarray method. This method of probe molecule deposition should be generally useful in the production of microarrays for label-free detection.

Quantitative proteomic analysis of human testis reveals system-wide molecular and cellular pathways associated with non-obstructive azoospermia.

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Alikhani, Mehdi; Mirzaei, Mehdi; Sabbaghian, Marjan; Parsamatin, Pouria; Karamzadeh, Razieh; Adib, Samane; Sodeifi, Niloofar; Gilani, Mohammad Ali Sadighi; Zabet-Moghaddam, Masoud; Parker, Lindsay; Wu, Yunqi; Gupta, Vivek; Haynes, Paul A; Gourabi, Hamid; Baharvand, Hossein; Salekdeh, Ghasem Hosseini

2017-06-06

Male infertility accounts for half of the infertility problems experienced by couples. Azoospermia, having no measurable level of sperm in seminal fluid, is one of the known conditions resulting in male infertility. In order to elucidate the complex molecular mechanisms causing male azoospermia, label-free quantitative shotgun proteomics was carried out on testicular tissue specimens from patients with obstructive azoospermia and non-obstructive azoospermia, including maturation arrest (MA) and Sertoli cell only syndrome (SCOS). The abundance of 520 proteins was significantly changed across three groups of samples. We were able to identify several functional biological pathways enriched in azoospermia samples and confirm selected differentially abundant proteins, using multiple histological methods. The results revealed that cell cycle and proteolysis, and RNA splicing were the most significant biological processes impaired by the substantial suppression of proteins related to the aforementioned categories in SCOS tissues. In the MA patient testes, generation of precursor metabolites and energy as well as oxidation-reduction were the most significantly altered processes. Novel candidate proteins identified in this study include key transcription factors, many of which have not previously been shown to be associated with azoospermia. Our findings can provide substantial insights into the molecular regulation of spermatogenesis and human reproduction. The obtained data showed a drastic suppression of proteins involved in spliceosome, cell cycle and proteasome proteins, as well as energy and metabolic production in Sertoli cell only syndrome testis tissue, and to a lesser extent in maturation arrest samples. Moreover, we identified new transcription factors that are highly down-regulated in SCOS and MA patients, thus helping to understand the molecular complexity of spermatogenesis in male infertility. Our findings provide novel candidate protein targets associated

Positional proteomics in the era of the human proteome project on the doorstep of precision medicine.

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Eckhard, Ulrich; Marino, Giada; Butler, Georgina S; Overall, Christopher M

2016-03-01

Proteolytic processing is a pervasive and irreversible post-translational modification that expands the protein universe by generating new proteoforms (protein isoforms). Unlike signal peptide or prodomain removal, protease-generated proteoforms can rarely be predicted from gene sequences. Positional proteomic techniques that enrich for N- or C-terminal peptides from proteomes are indispensable for a comprehensive understanding of a protein's function in biological environments since protease cleavage frequently results in altered protein activity and localization. Proteases often process other proteases and protease inhibitors which perturbs proteolytic networks and potentiates the initial cleavage event to affect other molecular networks and cellular processes in physiological and pathological conditions. This review is aimed at researchers with a keen interest in state of the art systems level positional proteomic approaches that: (i) enable the study of complex protease-protease, protease-inhibitor and protease-substrate crosstalk and networks; (ii) allow the identification of proteolytic signatures as candidate disease biomarkers; and (iii) are expected to fill the Human Proteome Project missing proteins gap. We predict that these methodologies will be an integral part of emerging precision medicine initiatives that aim to customize healthcare, converting reactive medicine into a personalized and proactive approach, improving clinical care and maximizing patient health and wellbeing, while decreasing health costs by eliminating ineffective therapies, trial-and-error prescribing, and adverse drug effects. Such initiatives require quantitative and functional proteome profiling and dynamic disease biomarkers in addition to current pharmacogenomics approaches. With proteases at the pathogenic center of many diseases, high-throughput protein termini identification techniques such as TAILS (Terminal Amine Isotopic Labeling of Substrates) and COFRADIC (COmbined

Mining the active proteome of Arabidopsis thaliana

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Renier A. L. Van Der Hoorn

2011-11-01

Full Text Available Assigning functions to the >30.000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied Activity-based Protein Profiling (ABPP. ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed of activities of 76 Arabidopsis proteins. These proteins represent over ten different protein classes that contain over 250 Arabidopsis proteins, including cysteine- serine- and metallo-proteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click-chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed novel protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities e.g. of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry and proteomics.

Asymmetric proteome equalization of the skeletal muscle proteome using a combinatorial hexapeptide library.

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Jenny Rivers

Full Text Available Immobilized combinatorial peptide libraries have been advocated as a strategy for equalization of the dynamic range of a typical proteome. The technology has been applied predominantly to blood plasma and other biological fluids such as urine, but has not been used extensively to address the issue of dynamic range in tissue samples. Here, we have applied the combinatorial library approach to the equalization of a tissue where there is also a dramatic asymmetry in the range of abundances of proteins; namely, the soluble fraction of skeletal muscle. We have applied QconCAT and label-free methodology to the quantification of the proteins that bind to the beads as the loading is progressively increased. Although some equalization is achieved, and the most abundant proteins no longer dominate the proteome analysis, at high protein loadings a new asymmetry of protein expression is reached, consistent with the formation of complex assembles of heat shock proteins, cytoskeletal elements and other proteins on the beads. Loading at different ionic strength values leads to capture of different subpopulations of proteins, but does not completely eliminate the bias in protein accumulation. These assemblies may impair the broader utility of combinatorial library approaches to the equalization of tissue proteomes. However, the asymmetry in equalization is manifest at either low and high ionic strength values but manipulation of the solvent conditions may extend the capacity of the method.

Proteome data from a host-pathogen interaction study with Staphylococcus aureus and human lung epithelial cells

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Kristin Surmann

2016-06-01

Full Text Available To simultaneously obtain proteome data of host and pathogen from an internalization experiment, human alveolar epithelial A549 cells were infected with Staphylococcus aureus HG001 which carried a plasmid (pMV158GFP encoding a continuously expressed green fluorescent protein (GFP. Samples were taken hourly between 1.5 h and 6.5 h post infection. By fluorescence activated cell sorting GFP-expressing bacteria could be enriched from host cell debris, but also infected host cells could be separated from those which did not carry bacteria after contact (exposed. Additionally, proteome data of A549 cells which were not exposed to S. aureus but underwent the same sample processing steps are provided as a control. Time-resolved changes in bacterial protein abundance were quantified in a label-free approach. Proteome adaptations of host cells were monitored by comparative analysis to a stable isotope labeled cell culture (SILAC standard. Proteins were extracted from the cells, digested proteolytically, measured by nanoLC–MS/MS, and subsequently identified by database search and then quantified. The data presented here are related to a previously published research article describing the interplay of S. aureus HG001 and human epithelial cells (Surmann et al., 2015 [1]. They have been deposited to the ProteomeXchange platform with the identifiers PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002384 for the S. aureus HG001 proteome dataset and PRIDE: http://www.ebi.ac.uk/pride/archive/projects/PXD002388 for the A549 proteome dataset.

Quantitative risk assessment of foods containing peanut advisory labeling.

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Remington, Benjamin C; Baumert, Joseph L; Marx, David B; Taylor, Steve L

2013-12-01

Foods with advisory labeling (i.e. "may contain") continue to be prevalent and the warning may be increasingly ignored by allergic consumers. We sought to determine the residual levels of peanut in various packaged foods bearing advisory labeling, compare similar data from 2005 and 2009, and determine any potential risk for peanut-allergic consumers. Of food products bearing advisory statements regarding peanut or products that had peanut listed as a minor ingredient, 8.6% and 37.5% contained detectable levels of peanut (>2.5 ppm whole peanut), respectively. Peanut-allergic individuals should be advised to avoid such products regardless of the wording of the advisory statement. Peanut was detected at similar rates and levels in products tested in both 2005 and 2009. Advisory labeled nutrition bars contained the highest levels of peanut and an additional market survey of 399 products was conducted. Probabilistic risk assessment showed the risk of a reaction to peanut-allergic consumers from advisory labeled nutrition bars was significant but brand-dependent. Peanut advisory labeling may be overused on some nutrition bars but prudently used on others. The probabilistic approach could provide the food industry with a quantitative method to assist with determining when advisory labeling is most appropriate. Copyright © 2013 Elsevier Ltd. All rights reserved.

Label-free and live cell imaging by interferometric scattering microscopy.

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Park, Jin-Sung; Lee, Il-Buem; Moon, Hyeon-Min; Joo, Jong-Hyeon; Kim, Kyoung-Hoon; Hong, Seok-Cheol; Cho, Minhaeng

2018-03-14

Despite recent remarkable advances in microscopic techniques, it still remains very challenging to directly observe the complex structure of cytoplasmic organelles in live cells without a fluorescent label. Here we report label-free and live-cell imaging of mammalian cell, Escherischia coli , and yeast, using interferometric scattering microscopy, which reveals the underlying structures of a variety of cytoplasmic organelles as well as the underside structure of the cells. The contact areas of the cells attached onto a glass substrate, e.g. , focal adhesions and filopodia, are clearly discernible. We also found a variety of fringe-like features in the cytoplasmic area, which may reflect the folded structures of cytoplasmic organelles. We thus anticipate that the label-free interferometric scattering microscopy can be used as a powerful tool to shed interferometric light on in vivo structures and dynamics of various intracellular phenomena.

Quantitative Analysis of Proteome Modulations in Alveolar Epithelial Type II Cells in Response to Pulmonary Aspergillus fumigatus Infection.

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Seddigh, Pegah; Bracht, Thilo; Molinier-Frenkel, Válerie; Castellano, Flavia; Kniemeyer, Olaf; Schuster, Marc; Weski, Juliane; Hasenberg, Anja; Kraus, Andreas; Poschet, Gernot; Hager, Thomas; Theegarten, Dirk; Opitz, Christiane A; Brakhage, Axel A; Sitek, Barbara; Hasenberg, Mike; Gunzer, Matthias

2017-12-01

The ubiquitous mold Aspergillus fumigatus threatens immunosuppressed patients as inducer of lethal invasive aspergillosis. A. fumigatus conidia are airborne and reach the alveoli, where they encounter alveolar epithelial cells (AEC). Previous studies reported the importance of the surfactant-producing AEC II during A. fumigatus infection via in vitro experiments using cell lines. We established a negative isolation protocol yielding untouched primary murine AEC II with a purity >90%, allowing ex vivo analyses of the cells, which encountered the mold in vivo By label-free proteome analysis of AEC II isolated from mice 24h after A. fumigatus or mock infection we quantified 2256 proteins and found 154 proteins to be significantly differentially abundant between both groups (ANOVA p value ≤ 0.01, ratio of means ≥1.5 or ≤0.67, quantified with ≥2 peptides). Most of these proteins were higher abundant in the infected condition and reflected a comprehensive activation of AEC II on interaction with A. fumigatus This was especially represented by proteins related to oxidative phosphorylation, hence energy production. However, the most strongly induced protein was the l-amino acid oxidase (LAAO) Interleukin 4 induced 1 (IL4I1) with a 42.9 fold higher abundance (ANOVA p value 2.91 -10 ). IL4I1 has previously been found in B cells, macrophages, dendritic cells and rare neurons. Increased IL4I1 abundance in AEC II was confirmed by qPCR, Western blot and immunohistology. Furthermore, A. fumigatus infected lungs showed high levels of IL4I1 metabolic products. Importantly, higher IL4I1 abundance was also confirmed in lung tissue from human aspergilloma. Because LAAO are key enzymes for bactericidal product generation, AEC II might actively participate in pathogen defense. We provide insights into proteome changes of primary AEC II thereby opening new avenues to analyze the molecular changes of this central lung cell on infectious threats. Data are available via Proteome

Quantitative Proteome Analysis Reveals Increased Content of Basement Membrane Proteins in Arteries from Patients with Type 2 Diabetes and Lower Levels among Metformin Users

DEFF Research Database (Denmark)

Rørdam Preil, Simone; Kristensen, Lars P; Beck, Hans C

2015-01-01

hypothesized that metformin intake influences the protein composition. METHODS AND RESULTS: -We analyzed non-atherosclerotic repair arteries gathered at coronary by-pass operations from 30 patients with type 2 diabetes, as well as from 30 age- and gender-matched non-diabetic individuals. Quantitative proteome......BACKGROUND: -The increased risk of cardiovascular diseases (CVD) in type 2 diabetes has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes and moreover...... analysis was done by iTRAQ-labelling and LC-MS/MS analysis on individual arterial samples. The amounts of the basement membrane (BM) components, alpha-1- and alpha-2- type IV collagen, gamma-1- and beta-2-laminin were significantly increased in patients with diabetes. Moreover, the expressions of basement...

Sperm-macrophage interaction in the mouse: a quantitative assay in vitro using 111indium oxine-labeled sperm

International Nuclear Information System (INIS)

Olive, D.L.; Weinberg, J.B.; Haney, A.F.

1987-01-01

The role of reproductive tract macrophages in contraception and reproductive failure has become widely recognized. However, in vitro analysis of sperm phagocytosis by macrophages has relied upon a semi-quantitative method of sperm counting that is of limited accuracy and reproducibility. We have developed an assay using murine sperm labeled with 111 indium oxine, and results indicate the labeling to be rapid and efficient. Incorporation of 111 indium into sperm increased the dose and sperm concentration and reached 90% maximal uptake after 15 min incubation, with maximal uptake occurring at 30 min. No decrease in sperm motility was noted with levels of oxine in excess of those required for significant labeling. Maximal labeling efficiency occurred in phosphate-buffered saline (PBS), with Dulbecco's modified Eagle's medium (DMEM) + 10% adult bovine serum (ABS) producing significantly less uptake. Label dissociation was detectable in PBS at room temperature, but at 37 degrees C in DMEM + 10% ABS, loss of label occurred at a rate of 23.5%/h. Addition of labeled sperm to murine macrophage monolayers under optimal conditions resulted in uptake of 111 indium by macrophages, while free label was unincorporated. Results indicated assay specificity for macrophage-limited uptake, with insignificant label uptake by nonphagocytic murine fibroblasts and better sensitivity than sperm counting. Macrophages from Bacillus Calmette-Guerin (BCG)-infected mice resulted in a decrease in sperm uptake. Female macrophages showed greater capacity for sperm uptake than those of the male mouse. These initial studies demonstrated the utility of this model system in enhancing the understanding of sperm-macrophage interaction in the female reproductive tract

The iron-responsive microsomal proteome of Aspergillus fumigatus.

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Moloney, Nicola M; Owens, Rebecca A; Meleady, Paula; Henry, Michael; Dolan, Stephen K; Mulvihill, Eoin; Clynes, Martin; Doyle, Sean

2016-03-16

Aspergillus fumigatus is an opportunistic fungal pathogen. Siderophore biosynthesis and iron acquisition are essential for virulence. Yet, limited data exist with respect to the adaptive nature of the fungal microsomal proteome under iron-limiting growth conditions, as encountered during host infection. Here, we demonstrate that under siderophore biosynthetic conditions--significantly elevated fusarinine C (FSC) and triacetylfusarinine C (TAFC) production (pproteome remodelling occurs. Specifically, a four-fold enrichment of transmembrane-containing proteins was observed with respect to whole cell lysates following ultracentrifugation-based microsomal extraction. Comparative label-free proteomic analysis of microsomal extracts, isolated following iron-replete and -deplete growth, identified 710 unique proteins. Scatterplot analysis (MaxQuant) demonstrated high correlation amongst biological replicates from each growth condition (Pearson correlation >0.96 within groups; biological replicates (n=4)). Quantitative and qualitative comparison revealed 231 proteins with a significant change in abundance between the iron-replete and iron-deplete conditions (pAspergillus fumigatus must acquire iron to facilitate growth and pathogenicity. Iron-chelating non-ribosomal peptides, termed siderophores, mediate iron uptake via membrane-localised transporter proteins. Here we demonstrate for the first time that growth of A. fumigatus under iron-deplete conditions, concomitant with siderophore biosynthesis, leads to an extensive remodelling of the microsomal proteome which includes significantly altered levels of 231 constituent proteins (96 increased and 135 decreased in abundance), many of which have not previously been localised to the microsome. We also demonstrate the first synthesis of a fluorescent version of fusarinine C, an extracellular A. fumigatus siderophore, and its uptake and localization under iron-restricted conditions. This infers the use of an A. fumigatus

Label-free detection of HIV-1 infected cells via integration of optical tweezers and photoluminescence spectroscopy

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Lugongolo, Masixole Yvonne; Ombinda-Lemboumba, Saturnin; Noto, Luyanda Lunga; Maaza, Malik; Mthunzi-Kufa, Patience

2018-02-01

The human immunodeficiency virus-1 (HIV-1) is currently detected using conventional qualitative and quantitative tests to determine the presence or absence of HIV in blood samples. However, the approach of these tests detects the presence of either viral antibodies or viral RNA that require labelling which may be costly, sophisticated and time consuming. A label-free approach of detecting the presence of HIV is therefore desirable. Of note optical tweezers can be coupled with other technologies including spectroscopy, which also investigates light-matter interactions. For example, coupling of optical tweezers with luminescence spectroscopy techniques has emerged as a powerful tool in biology for micro-manipulation, detection and analysis of individual cells. Integration of optical techniques has enabled studying biological particles in a label-free manner, whilst detecting functional groups and other essential molecules within mixed populations of cells. In the current study, an optical trapping system coupled to luminescence spectroscopy was utilised to detect the presence of HIV infection in TZM-bl cells in vitro. This was performed by infecting TZM-bl cells with the ZM53 HIV-1 pseudovirus, and incubating them for 48 hours prior analysis. The differences between infected and uninfected cells were thereafter displayed as shown by the spectrographs obtained. Combination of these two techniques has a potential in the field of infectious disease diagnostics.

Accurate label-free reaction kinetics determination using initial rate heat measurements

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Ebrahimi, Kourosh Honarmand; Hagedoorn, Peter-Leon; Jacobs, Denise; Hagen, Wilfred R.

2015-01-01

Accurate label-free methods or assays to obtain the initial reaction rates have significant importance in fundamental studies of enzymes and in application-oriented high throughput screening of enzyme activity. Here we introduce a label-free approach for obtaining initial rates of enzyme activity from heat measurements, which we name initial rate calorimetry (IrCal). This approach is based on our new finding that the data recorded by isothermal titration calorimetry for the early stages of a reaction, which have been widely ignored, are correlated to the initial rates. Application of the IrCal approach to various enzymes led to accurate enzyme kinetics parameters as compared to spectroscopic methods and enabled enzyme kinetic studies with natural substrate, e.g. proteases with protein substrates. Because heat is a label-free property of almost all reactions, the IrCal approach holds promise in fundamental studies of various enzymes and in use of calorimetry for high throughput screening of enzyme activity. PMID:26574737

Dataset for the proteomic inventory and quantitative analysis of the breast cancer hypoxic secretome associated with osteotropism

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Thomas R. Cox

2015-12-01

Full Text Available The cancer secretome includes all of the macromolecules secreted by cells into their microenvironment. Cancer cell secretomes are significantly different to that of normal cells reflecting the changes that normal cells have undergone during their transition to malignancy. More importantly, cancer secretomes are known to be active mediators of both local and distant host cells and play an important role in the progression and dissemination of cancer. Here we have quantitatively profiled both the composition of breast cancer secretomes associated with osteotropism, and their modulation under normoxic and hypoxic conditions. We detect and quantify 162 secretome proteins across all conditions which show differential hypoxic induction and association with osteotropism. Mass Spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000397 and the complete proteomic, bioinformatic and biological analyses are reported in Cox et al. (2015 [1].

Effects of Three Commonly-used Diuretics on the Urinary Proteome

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Xundou Li

2014-06-01

Full Text Available Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including diuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. Here, we evaluated the effects of three commonly-used diuretics (furosemide, F; hydrochlorothiazide, H; and spirolactone, S on the urinary proteome in rats. Urine samples were collected before and after intragastric administration of diuretics at therapeutic doses and the proteomes were analyzed using label-free liquid chromatography–tandem mass spectrometry (LC–MS/MS. Based on the criteria of P ⩽ 0.05, a fold change ⩾2, a spectral count ⩾5, and false positive rate (FDR ⩽1%, 14 proteins (seven for F, five for H, and two for S were identified by Progenesis LC–MS. The human orthologs of most of these 14 proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics.

Effects of three commonly-used diuretics on the urinary proteome.

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Li, Xundou; Zhao, Mindi; Li, Menglin; Jia, Lulu; Gao, Youhe

2014-06-01

Biomarker is the measurable change associated with a physiological or pathophysiological process. Unlike blood which has mechanisms to keep the internal environment homeostatic, urine is more likely to reflect changes of the body. As a result, urine is likely to be a better biomarker source than blood. However, since the urinary proteome is affected by many factors, including diuretics, careful evaluation of those effects is necessary if urinary proteomics is used for biomarker discovery. Here, we evaluated the effects of three commonly-used diuretics (furosemide, F; hydrochlorothiazide, H; and spirolactone, S) on the urinary proteome in rats. Urine samples were collected before and after intragastric administration of diuretics at therapeutic doses and the proteomes were analyzed using label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS). Based on the criteria of P≤0.05, a fold change ≥2, a spectral count ≥5, and false positive rate (FDR) ≤1%, 14 proteins (seven for F, five for H, and two for S) were identified by Progenesis LC-MS. The human orthologs of most of these 14 proteins are stable in the healthy human urinary proteome, and ten of them are reported as disease biomarkers. Thus, our results suggest that the effects of diuretics deserve more attention in future urinary protein biomarker studies. Moreover, the distinct effects of diuretics on the urinary proteome may provide clues to the mechanisms of diuretics. Copyright © 2014. Production and hosting by Elsevier Ltd.

Comprehensive profiling of proteome changes upon sequential deletion of deubiquitylating enzymes

DEFF Research Database (Denmark)

Poulsen, Jon W; Madsen, Christian Toft; Young, Clifford

2012-01-01

Deubiquitylating enzymes (DUBs) are a large group of proteases that regulate ubiquitin-dependent metabolic pathways by cleaving ubiquitin-protein bonds. Here we present a global study aimed at elucidating the effects DUBs have on protein abundance changes in eukaryotic cells. To this end we compare...... wild-type Saccharomyces cerevisiae to 20 DUB knock-out strains using quantitative proteomics to measure proteome-wide expression of isotope labeled proteins, and analyze the data in the context of known transcription-factor regulatory networks. Overall we find that protein abundances differ widely...... between individual deletion strains, demonstrating that removing just a single component from the complex ubiquitin system causes major changes in cellular protein expression. The outcome of our analysis confirms many of the known biological roles for characterized DUBs such as Ubp3p and Ubp8p, and we...

Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis.

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Bracht, Thilo; Schweinsberg, Vincent; Trippler, Martin; Kohl, Michael; Ahrens, Maike; Padden, Juliet; Naboulsi, Wael; Barkovits, Katalin; Megger, Dominik A; Eisenacher, Martin; Borchers, Christoph H; Schlaak, Jörg F; Hoffmann, Andreas-Claudius; Weber, Frank; Baba, Hideo A; Meyer, Helmut E; Sitek, Barbara

2015-05-01

Hepatic fibrosis and cirrhosis are major health problems worldwide. Until now, highly invasive biopsy remains the diagnostic gold standard despite many disadvantages. To develop noninvasive diagnostic assays for the assessment of liver fibrosis, it is urgently necessary to identify molecules that are robustly expressed in association with the disease. We analyzed biopsied tissue samples from 95 patients with HBV/HCV-associated hepatic fibrosis using three different quantification methods. We performed a label-free proteomics discovery study to identify novel disease-associated proteins using a subset of the cohort (n = 27). Subsequently, gene expression data from all available clinical samples were analyzed (n = 77). Finally, we performed a targeted proteomics approach, multiple reaction monitoring (MRM), to verify the disease-associated expression in samples independent from the discovery approach (n = 68). We identified fibulin-5 (FBLN5) as a novel protein expressed in relation to hepatic fibrosis. Furthermore, we confirmed the altered expression of microfibril-associated glycoprotein 4 (MFAP4), lumican (LUM), and collagen alpha-1(XIV) chain (COL14A1) in association to hepatic fibrosis. To our knowledge, no tissue-based quantitative proteomics study for hepatic fibrosis has been performed using a cohort of comparable size. By this means, we add substantial evidence for the disease-related expression of the proteins examined in this study.

A knowledge-based T2-statistic to perform pathway analysis for quantitative proteomic data.

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Lai, En-Yu; Chen, Yi-Hau; Wu, Kun-Pin

2017-06-01

Approaches to identify significant pathways from high-throughput quantitative data have been developed in recent years. Still, the analysis of proteomic data stays difficult because of limited sample size. This limitation also leads to the practice of using a competitive null as common approach; which fundamentally implies genes or proteins as independent units. The independent assumption ignores the associations among biomolecules with similar functions or cellular localization, as well as the interactions among them manifested as changes in expression ratios. Consequently, these methods often underestimate the associations among biomolecules and cause false positives in practice. Some studies incorporate the sample covariance matrix into the calculation to address this issue. However, sample covariance may not be a precise estimation if the sample size is very limited, which is usually the case for the data produced by mass spectrometry. In this study, we introduce a multivariate test under a self-contained null to perform pathway analysis for quantitative proteomic data. The covariance matrix used in the test statistic is constructed by the confidence scores retrieved from the STRING database or the HitPredict database. We also design an integrating procedure to retain pathways of sufficient evidence as a pathway group. The performance of the proposed T2-statistic is demonstrated using five published experimental datasets: the T-cell activation, the cAMP/PKA signaling, the myoblast differentiation, and the effect of dasatinib on the BCR-ABL pathway are proteomic datasets produced by mass spectrometry; and the protective effect of myocilin via the MAPK signaling pathway is a gene expression dataset of limited sample size. Compared with other popular statistics, the proposed T2-statistic yields more accurate descriptions in agreement with the discussion of the original publication. We implemented the T2-statistic into an R package T2GA, which is available at https

Attached and planktonic Listeria monocytogenes global proteomic responses and associated influence of strain genetics and temperature.

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Mata, Marcia M; da Silva, Wladimir P; Wilson, Richard; Lowe, Edwin; Bowman, John P

2015-02-06

Contamination of industrial and domestic food usage environments by the attachement of bacterial food-borne pathogen Listeria monocytogenes has public health and economic implications. Comprehensive proteomics experiments using label-free liquid chromatography/tandem mass spectrometry were used to compare the proteomes of two different L. monocytogenes strains (Siliken_1/2c and F2365_4b), which show very different capacities to attach to surfaces. Growth temperature and strain type were highly influential on the proteomes in both attached and planktonic cells. On the basis of the proteomic data, it is highly unlikely that specific surface proteins play a direct role in adherence to inanimate surfaces. Instead, strain-dependent responses related to cell envelope polymer biosynthesis and stress response regulation likely contribute to a different ability to attach and also to survive external stressors. Collectively, the divergent proteome-level responses observed define strain- and growth-temperature-dependent differences relevant to attachment efficacy, highlight relevant proteins involved in stress protection in attached cells, and suggest that strain differences and growth conditions are important in relation to environmental persistence.

Label-Free Imaging and Biochemical Characterization of Bovine Sperm Cells

Science.gov (United States)

Ferrara, Maria Antonietta; Di Caprio, Giuseppe; Managò, Stefano; De Angelis, Annalisa; Sirleto, Luigi; Coppola, Giuseppe; De Luca, Anna Chiara

2015-01-01

A full label-free morphological and biochemical characterization is desirable to select spermatozoa during preparation for artificial insemination. In order to study these fundamental parameters, we take advantage of two attractive techniques: digital holography (DH) and Raman spectroscopy (RS). DH presents new opportunities for studying morphological aspect of cells and tissues non-invasively, quantitatively and without the need for staining or tagging, while RS is a very specific technique allowing the biochemical analysis of cellular components with a spatial resolution in the sub-micrometer range. In this paper, morphological and biochemical bovine sperm cell alterations were studied using these techniques. In addition, a complementary DH and RS study was performed to identify X- and Y-chromosome-bearing sperm cells. We demonstrate that the two techniques together are a powerful and highly efficient tool elucidating some important criterions for sperm morphological selection and sex-identification, overcoming many of the limitations associated with existing protocols. PMID:25836358

A Proteomic Approach for Plasma Biomarker Discovery with iTRAQ Labelling and OFFGEL Fractionation

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Emilie Ernoult

2010-01-01

Full Text Available Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma “signatures,” which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 μg of plasma proteins.

A proteomic approach for plasma biomarker discovery with iTRAQ labelling and OFFGEL fractionation.

Science.gov (United States)

Ernoult, Emilie; Bourreau, Anthony; Gamelin, Erick; Guette, Catherine

2010-01-01

Human blood plasma contains a plethora of proteins, encompassing not only proteins that have plasma-based functionalities, but also possibly every other form of low concentrated human proteins. As it circulates through the tissues, the plasma picks up proteins that are released from their origin due to physiological events such as tissue remodeling and cell death. Specific disease processes or tumors are often characterized by plasma "signatures," which may become obvious via changes in the plasma proteome profile, for example, through over expression of proteins. However, the wide dynamic range of proteins present in plasma makes their analysis very challenging, because high-abundance proteins tend to mask those of lower abundance. In the present study, we used a strategy combining iTRAQ as a reagent which improved the peptide ionization and peptide OFFGEL fractionation that has already been shown, in our previous research, to improve the proteome coverage of cellular extracts. Two prefractioning methods were compared: immunodepletion and a bead-based library of combinatorial hexapeptide technology. Our data suggested that both methods were complementary, with regard to the number of identified proteins. iTRAQ labelling, in association with OFFGEL fractionation, allowed more than 300 different proteins to be characterized from 400 microg of plasma proteins.

Quantitative Proteomic Profiling the Molecular Signatures of Annexin A5 in Lung Squamous Carcinoma Cells.

Science.gov (United States)

Sun, Bing; Bai, Yuxin; Zhang, Liyuan; Gong, Linlin; Qi, Xiaoyu; Li, Huizhen; Wang, Faming; Chi, Xinming; Jiang, Yulin; Shao, Shujuan

Lung cancer remains the leading cancer killer around the world. It's crucial to identify newer mechanism-based targets to effectively manage lung cancer. Annexin A5 (ANXA5) is a protein kinase C inhibitory protein and calcium dependent phospholipid-binding protein, which may act as an endogenous regulator of various pathophysiological processes. However, its molecular mechanism in lung cancer remains poorly understood. This study was designed to determine the mechanism of ANXA5 in lung cancer with a hope to obtain useful information to provide a new therapeutic target. We used a stable isotope dimethyl labeling based quantitative proteomic method to identify differentially expressed proteins in NSCLC cell lines after ANXA5 transfection. Out of 314 proteins, we identified 26 and 44 proteins that were down- and up-regulated upon ANXA5 modulation, respectively. The IPA analysis revealed that glycolysis and gluconeogenesis were the predominant pathways modulated by ANXA5. Multiple central nodes, namely HSPA5, FN1, PDIA6, ENO1, ALDOA, JUP and KRT6A appeared to occupy regulatory nodes in the protein-protein networks upon ANXA5 modulation. Taken together, ANXA5 appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of ANXA5 as a potential target in lung cancer. This study might provide a new insight into the mechanism of ANXA5 in lung cancer.

Quantitative Proteomic Profiling the Molecular Signatures of Annexin A5 in Lung Squamous Carcinoma Cells.

Directory of Open Access Journals (Sweden)

Bing Sun

Full Text Available Lung cancer remains the leading cancer killer around the world. It's crucial to identify newer mechanism-based targets to effectively manage lung cancer. Annexin A5 (ANXA5 is a protein kinase C inhibitory protein and calcium dependent phospholipid-binding protein, which may act as an endogenous regulator of various pathophysiological processes. However, its molecular mechanism in lung cancer remains poorly understood. This study was designed to determine the mechanism of ANXA5 in lung cancer with a hope to obtain useful information to provide a new therapeutic target. We used a stable isotope dimethyl labeling based quantitative proteomic method to identify differentially expressed proteins in NSCLC cell lines after ANXA5 transfection. Out of 314 proteins, we identified 26 and 44 proteins that were down- and up-regulated upon ANXA5 modulation, respectively. The IPA analysis revealed that glycolysis and gluconeogenesis were the predominant pathways modulated by ANXA5. Multiple central nodes, namely HSPA5, FN1, PDIA6, ENO1, ALDOA, JUP and KRT6A appeared to occupy regulatory nodes in the protein-protein networks upon ANXA5 modulation. Taken together, ANXA5 appears to have pleotropic effects, as it modulates multiple key signaling pathways, supporting the potential usefulness of ANXA5 as a potential target in lung cancer. This study might provide a new insight into the mechanism of ANXA5 in lung cancer.

The Seed Proteome Web Portal

Directory of Open Access Journals (Sweden)

Marc eGalland

2012-06-01

Full Text Available The Seed Proteome Web Portal (SPWP; http://www.seedproteome.com/ gives access to information both on quantitative seed proteomic data and on seed-related protocols. Firstly, the SPWP provides access to the 475 different Arabidopsis seed proteins annotated from 2 dimensional electrophoresis (2DE maps. Quantitative data are available for each protein according to their accumulation profile during the germination process. These proteins can be retrieved either in list format or directly on scanned 2DE maps. These proteomic data reveal that 40% of seed proteins maintain a stable abundance over germination, up to radicle protrusion. During sensu stricto germination (24 h upon imbibition about 50% of the proteins display quantitative variations, exhibiting an increased abundance (35% or a decreasing abundance (15%. Moreover, during radicle protrusion (24 h to 48 h upon imbibition, 41% proteins display quantitative variations with an increased (23% or a decreasing abundance (18%. In addition, an analysis of the seed proteome revealed the importance of protein post-translational modifications as demonstrated by the poor correlation (r2 = 0.29 between the theoretical (predicted from Arabidopsis genome and the observed protein isoelectric points. Secondly, the SPWP is a relevant technical resource for protocols specifically dedicated to Arabidopsis seed proteome studies. Concerning 2D electrophoresis, the user can find efficient procedures for sample preparation, electrophoresis coupled with gel analysis and protein identification by mass spectrometry, which we have routinely used during the last 12 years. Particular applications such as the detection of oxidized proteins or de novo synthetized proteins radiolabeled by [35S]-methionine are also given in great details. Future developments of this portal will include proteomic data from studies such as dormancy release and protein turnover through de novo protein synthesis analyses during germination.

Differential Labeling of Free and Disulfide-Bound Thiol Functions in Proteins

NARCIS (Netherlands)

Seiwert, B.; Hayen, H.; Karst, U.

2008-01-01

A method for the simultaneous determination of the number of free cysteine groups and disulfide-bound cysteine groups in proteins has been developed based on the sequential labeling of free and bound thiol functionalities with two ferrocene-based maleimide reagents. Liquid

Quantitative proteomics and dynamic imaging of the nucleolus reveal distinct responses to UV and ionizing radiation.

Science.gov (United States)

Moore, Henna M; Bai, Baoyan; Boisvert, François-Michel; Latonen, Leena; Rantanen, Ville; Simpson, Jeremy C; Pepperkok, Rainer; Lamond, Angus I; Laiho, Marikki

2011-10-01

The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

Fusarium oxysporum mediates systems metabolic reprogramming of chickpea roots as revealed by a combination of proteomics and metabolomics.

Science.gov (United States)

Kumar, Yashwant; Zhang, Limin; Panigrahi, Priyabrata; Dholakia, Bhushan B; Dewangan, Veena; Chavan, Sachin G; Kunjir, Shrikant M; Wu, Xiangyu; Li, Ning; Rajmohanan, Pattuparambil R; Kadoo, Narendra Y; Giri, Ashok P; Tang, Huiru; Gupta, Vidya S

2016-07-01

Molecular changes elicited by plants in response to fungal attack and how this affects plant-pathogen interaction, including susceptibility or resistance, remain elusive. We studied the dynamics in root metabolism during compatible and incompatible interactions between chickpea and Fusarium oxysporum f. sp. ciceri (Foc), using quantitative label-free proteomics and NMR-based metabolomics. Results demonstrated differential expression of proteins and metabolites upon Foc inoculations in the resistant plants compared with the susceptible ones. Additionally, expression analysis of candidate genes supported the proteomic and metabolic variations in the chickpea roots upon Foc inoculation. In particular, we found that the resistant plants revealed significant increase in the carbon and nitrogen metabolism; generation of reactive oxygen species (ROS), lignification and phytoalexins. The levels of some of the pathogenesis-related proteins were significantly higher upon Foc inoculation in the resistant plant. Interestingly, results also exhibited the crucial role of altered Yang cycle, which contributed in different methylation reactions and unfolded protein response in the chickpea roots against Foc. Overall, the observed modulations in the metabolic flux as outcome of several orchestrated molecular events are determinant of plant's role in chickpea-Foc interactions. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

PyQuant: A Versatile Framework for Analysis of Quantitative Mass Spectrometry Data.

Science.gov (United States)

Mitchell, Christopher J; Kim, Min-Sik; Na, Chan Hyun; Pandey, Akhilesh

2016-08-01

Quantitative mass spectrometry data necessitates an analytical pipeline that captures the accuracy and comprehensiveness of the experiments. Currently, data analysis is often coupled to specific software packages, which restricts the analysis to a given workflow and precludes a more thorough characterization of the data by other complementary tools. To address this, we have developed PyQuant, a cross-platform mass spectrometry data quantification application that is compatible with existing frameworks and can be used as a stand-alone quantification tool. PyQuant supports most types of quantitative mass spectrometry data including SILAC, NeuCode, (15)N, (13)C, or (18)O and chemical methods such as iTRAQ or TMT and provides the option of adding custom labeling strategies. In addition, PyQuant can perform specialized analyses such as quantifying isotopically labeled samples where the label has been metabolized into other amino acids and targeted quantification of selected ions independent of spectral assignment. PyQuant is capable of quantifying search results from popular proteomic frameworks such as MaxQuant, Proteome Discoverer, and the Trans-Proteomic Pipeline in addition to several standalone search engines. We have found that PyQuant routinely quantifies a greater proportion of spectral assignments, with increases ranging from 25-45% in this study. Finally, PyQuant is capable of complementing spectral assignments between replicates to quantify ions missed because of lack of MS/MS fragmentation or that were omitted because of issues such as spectra quality or false discovery rates. This results in an increase of biologically useful data available for interpretation. In summary, PyQuant is a flexible mass spectrometry data quantification platform that is capable of interfacing with a variety of existing formats and is highly customizable, which permits easy configuration for custom analysis. © 2016 by The American Society for Biochemistry and Molecular Biology

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

Energy Technology Data Exchange (ETDEWEB)

Liu, Xia [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Fifth People' s Hospital of Shanghai, School of Medicine, Fudan University, Shanghai, 200240 (China); Zhao, Libo [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Department of Neurology, The Third People' s Hospital of Chongqing, 400014 (China); Yang, Yongtao [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Bode, Liv [Bornavirus Research Group affiliated to the Free University of Berlin, Berlin (Germany); Huang, Hua [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Liu, Chengyu [Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); Huang, Rongzhong [Department of Rehabilitative Medicine, The Second Affiliated Hospital of Chongqing Medical University, Chongqing, 400010 (China); Zhang, Liang [Department of Neurology, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016 (China); Chongqing Key Laboratory of Neurobiology, Chongqing Medical University, Chongqing, 400016 (China); Institute of Neuroscience, Chongqing Medical University, Chongqing, 400016 (China); and others

2014-09-15

Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs.

Human borna disease virus infection impacts host proteome and histone lysine acetylation in human oligodendroglia cells

International Nuclear Information System (INIS)

Liu, Xia; Zhao, Libo; Yang, Yongtao; Bode, Liv; Huang, Hua; Liu, Chengyu; Huang, Rongzhong; Zhang, Liang

2014-01-01

Background: Borna disease virus (BDV) replicates in the nucleus and establishes persistent infections in mammalian hosts. A human BDV strain was used to address the first time, how BDV infection impacts the proteome and histone lysine acetylation (Kac) of human oligodendroglial (OL) cells, thus allowing a better understanding of infection-driven pathophysiology in vitro. Methods: Proteome and histone lysine acetylation were profiled through stable isotope labeling for cell culture (SILAC)-based quantitative proteomics. The quantifiable proteome was annotated using bioinformatics. Histone acetylation changes were validated by biochemistry assays. Results: Post BDV infection, 4383 quantifiable differential proteins were identified and functionally annotated to metabolism pathways, immune response, DNA replication, DNA repair, and transcriptional regulation. Sixteen of the thirty identified Kac sites in core histones presented altered acetylation levels post infection. Conclusions: BDV infection using a human strain impacted the whole proteome and histone lysine acetylation in OL cells. - Highlights: • A human strain of BDV (BDV Hu-H1) was used to infect human oligodendroglial cells (OL cells). • This study is the first to reveal the host proteomic and histone Kac profiles in BDV-infected OL cells. • BDV infection affected the expression of many transcription factors and several HATs and HDACs

Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese.

Science.gov (United States)

Delgado, Josué; Owens, Rebecca A; Doyle, Sean; Núñez, Félix; Asensio, Miguel A

2017-09-01

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl 2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl 2 -incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl 2 . Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products. Copyright © 2017 Elsevier Ltd. All rights reserved.

Quantitative, high-resolution proteomics for data-driven systems biology

DEFF Research Database (Denmark)

Cox, J.; Mann, M.

2011-01-01

Systems biology requires comprehensive data at all molecular levels. Mass spectrometry (MS)-based proteomics has emerged as a powerful and universal method for the global measurement of proteins. In the most widespread format, it uses liquid chromatography (LC) coupled to high-resolution tandem...... primary structure of proteins including posttranslational modifications, to localize proteins to organelles, and to determine protein interactions. Here, we describe the principles of analysis and the areas of biology where proteomics can make unique contributions. The large-scale nature of proteomics...... data and its high accuracy pose special opportunities as well as challenges in systems biology that have been largely untapped so far....

Quantitation of thrombogenicity of hemodialyzer with technetium-99m and indium-111 labeled platelets

Energy Technology Data Exchange (ETDEWEB)

Dewanjee, M.K.; Kapadvanjwala, Mansoor; Ruzius, Kees; Serafini, A.N.; Zilleruelo, G.E.; Sfakianakis, G.N. (Miami Univ., FL (United States). School of Medicine Althin CD-Medical Inc., Miami Lakes, FL (United States))

1993-07-01

The platelet thromobogenicity of a hemodialyzer was quantified with [sup 99m]Tc- and [sup 111]In-labeled platelets. The platelets collected from blood of Beagle dogs, Yorkshire pigs and human volunteers were labeled with [sup 111]in-tropolone (detergent-free) and [sup 99m]Tc-HMPAO. Hemodialysis was performed with a hollow-fiber dialyzer (HFD) in a flow-loop, the temperature of which was maintained at 37[sup o]C, with flow-rates of 7, 150 and 270 mL/min; after dialysis, the HFD radioactivity was measured with an ionization chamber and imaged with a [gamma]-camera. The radioactivity of samples of hollow-fibers taken from the top, middle and bottom of the dialyzer was determined with a [gamma]-counter. The mean values of hemodialyzer-adherent platelet radioactivity were calculated for both radionuclides. The canine platelets were found to be more thrombogenic than porcine and human platelets. The adhesivity of porcine platelets to the biomaterial (cellulose-acetate) of the dialyzer approximated that of human platelets. The [sup 99m]Tc label underestimated the thrombus formation (P < 0.01 ). The dynamic processes of thrombosis and embolization from the hemodialyzer resulted in the large standard deviations around the mean values of the adherent thrombus. In spite of this limitation of the dynamic pathology, the quantitation of comparative throbogenicity with [sup 111]In- and [sup 99m]Tc-labeled platelets suggests that both radionuclides could be used for measurement of device-induced thrombogenicity and may provide an estimation of prosthesis-induced thrombogenicity of human platelets from animal studies. (Author).

Differential effect of TGFβ on the proteome of cancer associated fibroblasts and cancer epithelial cells in a co-culture approach - a short report.

Science.gov (United States)

Koczorowska, Maria Magdalena; Friedemann, Charlotte; Geiger, Klaus; Follo, Marie; Biniossek, Martin Lothar; Schilling, Oliver

2017-12-01

Solid tumors contain various components that together form the tumor microenvironment. Cancer associated fibroblasts (CAFs) are capable of secreting and responding to signaling molecules and growth factors. Due to their role in tumor development, CAFs are considered as potential therapeutic targets. A prominent tumor-associated signaling molecule is transforming growth factor β (TGFβ), an inducer of epithelial-to-mesenchymal transition (EMT). The differential action of TGFβ on CAFs and ETCs (epithelial tumor cells) has recently gained interest. Here, we aimed to investigate the effects of TGFβ on CAFs and ETCs at the proteomic level. We established a 2D co-culture system of differentially fluorescently labeled CAFs and ETCs and stimulated this co-culture system with TGFβ. The respective cell types were separated using FACS and subjected to quantitative analyses of individual proteomes using mass spectrometry. We found that TGFβ treatment had a strong impact on the proteome composition of CAFs, whereas ETCs responded only marginally to TGFβ. Quantitative proteomic analyses of the different cell types revealed up-regulation of extracellular matrix (ECM) proteins in TGFβ treated CAFs. In addition, we found that the TGFβ treated CAFs exhibited increased N-cadherin levels. From our data we conclude that CAFs respond to TGFβ treatment by changing their proteome composition, while ETCs appear to be rather resilient.

Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

Science.gov (United States)

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-03-01

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery.

Response of Human Osteoblast to n-HA/PEEK—Quantitative Proteomic Study of Bio-effects of Nano-Hydroxyapatite Composite

Science.gov (United States)

Zhao, Minzhi; Li, Haiyun; Liu, Xiaochen; Wei, Jie; Ji, Jianguo; Yang, Shu; Hu, Zhiyuan; Wei, Shicheng

2016-01-01

Nano-sized hydroxyapatite (n-HA) is considered as a bio-active material, which is often mixed into bone implant material, polyetheretherketone (PEEK). To reveal the global protein expression modulations of osteoblast in response to direct contact with the PEEK composite containing high level (40%) nano-sized hydroxyapatite (n-HA/PEEK) and explain its comprehensive bio-effects, quantitative proteomic analysis was conducted on human osteoblast-like cells MG-63 cultured on n-HA/PEEK in comparison with pure PEEK. Results from quantitative proteomic analysis showed that the most enriched categories in the up-regulated proteins were related to calcium ion processes and associated functions while the most enriched categories in the down-regulated proteins were related to RNA process. This enhanced our understanding to the molecular mechanism of the promotion of the cell adhesion and differentiation with the inhibition of the cell proliferation on n-HA/PEEK composite. It also exhibited that although the calcium ion level of incubate environment hadn’t increased, merely the calcium fixed on the surface of material had influence to intracellular calcium related processes, which was also reflect by the higher intracellular Ca2+ concentration of n-HA/PEEK. This study could lead to more comprehensive cognition to the versatile biocompatibility of composite materials. It further proves that proteomics is useful in new bio-effect discovery. PMID:26956660

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.

Science.gov (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

2016-08-11

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

Proteomics Insights into the Biomass Hydrolysis Potentials of a Hypercellulolytic Fungus Penicillium funiculosum.

Science.gov (United States)

Ogunmolu, Funso Emmanuel; Kaur, Inderjeet; Gupta, Mayank; Bashir, Zeenat; Pasari, Nandita; Yazdani, Syed Shams

2015-10-02

The quest for cheaper and better enzymes needed for the efficient hydrolysis of lignocellulosic biomass has placed filamentous fungi in the limelight for bioprospecting research. In our search for efficient biomass degraders, we identified a strain of Penicillium funiculosum whose secretome demonstrates high saccharification capabilities. Our probe into the secretome of the fungus through qualitative and label-free quantitative mass spectrometry based proteomics studies revealed a high abundance of inducible CAZymes and several nonhydrolytic accessory proteins. The preferential association of these proteins and the attending differential biomass hydrolysis gives an insight into their interactions and clues about possible roles of novel hydrolytic and nonhydrolytic proteins in the synergistic deconstruction of lignocellulosic biomass. Our study thus provides the first comprehensive insight into the repertoire of proteins present in a high-performing secretome of a hypercellulolytic Penicillium funiculosum, their relative abundance in the secretome, and the interaction dynamics of the various protein groups in the secretome. The gleanings from the stoichiometry of these interactions hold a prospect as templates in the design of cost-effective synthetic cocktails for the optimal hydrolysis of biomass.

Quantitative proteomics of the tonoplast reveals a role for glycolytic enzymes in salt tolerance.

Science.gov (United States)

Barkla, Bronwyn J; Vera-Estrella, Rosario; Hernández-Coronado, Marcela; Pantoja, Omar

2009-12-01

To examine the role of the tonoplast in plant salt tolerance and identify proteins involved in the regulation of transporters for vacuolar Na(+) sequestration, we exploited a targeted quantitative proteomics approach. Two-dimensional differential in-gel electrophoresis analysis of free flow zonal electrophoresis separated tonoplast fractions from control, and salt-treated Mesembryanthemum crystallinum plants revealed the membrane association of glycolytic enzymes aldolase and enolase, along with subunits of the vacuolar H(+)-ATPase V-ATPase. Protein blot analysis confirmed coordinated salt regulation of these proteins, and chaotrope treatment indicated a strong tonoplast association. Reciprocal coimmunoprecipitation studies revealed that the glycolytic enzymes interacted with the V-ATPase subunit B VHA-B, and aldolase was shown to stimulate V-ATPase activity in vitro by increasing the affinity for ATP. To investigate a physiological role for this association, the Arabidopsis thaliana cytoplasmic enolase mutant, los2, was characterized. These plants were salt sensitive, and there was a specific reduction in enolase abundance in the tonoplast from salt-treated plants. Moreover, tonoplast isolated from mutant plants showed an impaired ability for aldolase stimulation of V-ATPase hydrolytic activity. The association of glycolytic proteins with the tonoplast may not only channel ATP to the V-ATPase, but also directly upregulate H(+)-pump activity.

Label-Free Alignment of Nonmagnetic Particles in a Small Uniform Magnetic Field.

Science.gov (United States)

Wang, Zhaomeng; Wang, Ying; Wu, Rui Ge; Wang, Z P; Ramanujan, R V

2018-01-01

Label-free manipulation of biological entities can minimize damage, increase viability and improve efficiency of subsequent analysis. Understanding the mechanism of interaction between magnetic and nonmagnetic particles in an inverse ferrofluid can provide a mechanism of label-free manipulation of such entities in a uniform magnetic field. The magnetic force, induced by relative magnetic susceptibility difference between nonmagnetic particles and surrounding magnetic particles as well as particle-particle interaction were studied. Label-free alignment of nonmagnetic particles can be achieved by higher magnetic field strength (Ba), smaller particle spacing (R), larger particle size (rp1), and higher relative magnetic permeability difference between particle and the surrounding fluid (Rμr). Rμr can be used to predict the direction of the magnetic force between both magnetic and nonmagnetic particles. A sandwich structure, containing alternate layers of magnetic and nonmagnetic particle chains, was studied. This work can be used for manipulation of nonmagnetic particles in lab-on-a-chip applications.

Proteomic analysis of blastema formation in regenerating axolotl limbs

Directory of Open Access Journals (Sweden)

Nye Holly LD

2009-11-01

Full Text Available Abstract Background Following amputation, urodele salamander limbs reprogram somatic cells to form a blastema that self-organizes into the missing limb parts to restore the structure and function of the limb. To help understand the molecular basis of blastema formation, we used quantitative label-free liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS-based methods to analyze changes in the proteome that occurred 1, 4 and 7 days post amputation (dpa through the mid-tibia/fibula of axolotl hind limbs. Results We identified 309 unique proteins with significant fold change relative to controls (0 dpa, representing 10 biological process categories: (1 signaling, (2 Ca2+ binding and translocation, (3 transcription, (4 translation, (5 cytoskeleton, (6 extracellular matrix (ECM, (7 metabolism, (8 cell protection, (9 degradation, and (10 cell cycle. In all, 43 proteins exhibited exceptionally high fold changes. Of these, the ecotropic viral integrative factor 5 (EVI5, a cell cycle-related oncoprotein that prevents cells from entering the mitotic phase of the cell cycle prematurely, was of special interest because its fold change was exceptionally high throughout blastema formation. Conclusion Our data were consistent with previous studies indicating the importance of inositol triphosphate and Ca2+ signaling in initiating the ECM and cytoskeletal remodeling characteristic of histolysis and cell dedifferentiation. In addition, the data suggested that blastema formation requires several mechanisms to avoid apoptosis, including reduced metabolism, differential regulation of proapoptotic and antiapoptotic proteins, and initiation of an unfolded protein response (UPR. Since there is virtually no mitosis during blastema formation, we propose that high levels of EVI5 function to arrest dedifferentiated cells somewhere in the G1/S/G2 phases of the cell cycle until they have accumulated under the wound epidermis and enter mitosis in response to

Refining comparative proteomics by spectral counting to account for shared peptides and multiple search engines.

Science.gov (United States)

Chen, Yao-Yi; Dasari, Surendra; Ma, Ze-Qiang; Vega-Montoto, Lorenzo J; Li, Ming; Tabb, David L

2012-09-01

Spectral counting has become a widely used approach for measuring and comparing protein abundance in label-free shotgun proteomics. However, when analyzing complex samples, the ambiguity of matching between peptides and proteins greatly affects the assessment of peptide and protein inventories, differentiation, and quantification. Meanwhile, the configuration of database searching algorithms that assign peptides to MS/MS spectra may produce different results in comparative proteomic analysis. Here, we present three strategies to improve comparative proteomics through spectral counting. We show that comparing spectral counts for peptide groups rather than for protein groups forestalls problems introduced by shared peptides. We demonstrate the advantage and flexibility of this new method in two datasets. We present four models to combine four popular search engines that lead to significant gains in spectral counting differentiation. Among these models, we demonstrate a powerful vote counting model that scales well for multiple search engines. We also show that semi-tryptic searching outperforms tryptic searching for comparative proteomics. Overall, these techniques considerably improve protein differentiation on the basis of spectral count tables.

Photonic Crystal Biosensor Chip for Label-Free Detection of Bacteria