[Show abstract][Hide abstract] ABSTRACT: Glioblastoma multiforme (GBM) is a highly invasive and aggressive brain tumor with an invariably poor prognosis. The overexpression of epidermal growth factor receptor (EGFR) is a primary influencer of invasion and proliferation in tumor cells and the constitutively active EGFRvIII mutant, found in 30-65% of GBMs, confers more aggressive invasion. To better understand how EGFR contributes to tumor aggressiveness, we investigated the effect of EGFR on the secreted levels of 65 rationally selected proteins involved in invasion. We employed selected reaction monitoring (SRM) targeted mass spectrometry using stable isotope labeled internal peptide standards to quantity proteins in the secretome from five GBM (U87) isogenic cell lines in which EGFR, EGFRvIII and/or PTEN were expressed. Our results show that cell lines with EGFR overexpression and constitutive EGFRvIII expression differ remarkably in the expression profiles for both secreted and intracellular signaling proteins, and alterations in EGFR signaling result in reproducible changes in concentrations of secreted proteins. Furthermore, the EGFRvIII-expressing mutant cell line secretes the majority of the selected invasion-promoting proteins at higher levels than other cell lines tested. Additionally, the intracellular and extracellular protein measurements indicate elevated oxidative stress in the EGFRvIII-expressing cell line. In conclusion, the results of our study demonstrate that EGFR signaling has a significant effect on the levels of secreted invasion-promoting proteins, likely contributing to the aggressiveness of GBM. Further characterization of these proteins may provide candidates for new therapeutic strategies and targets as well as biomarkers for this aggressive disease.
[Show abstract][Hide abstract] ABSTRACT: Reversible protein phosphorylation determines growth and adaptive decisions in Mycobacterium tuberculosis (Mtb). At least 11 two-component systems and 11 Ser/Thr protein kinases (STPKs) mediate phosphorylation on Asp, His, Ser, and Thr. In contrast, protein phosphorylation on Tyr has not been described previously in Mtb. Here, using a combination of phospho-enrichment and highly sensitive mass spectrometry, we show extensive protein Tyr phosphorylation of diverse Mtb proteins, including STPKs. Several STPKs function as dual-specificity kinases that phosphorylate Tyr in cis and in trans, suggesting that dual-specificity kinases have a major role in bacterial phospho-signaling. Mutation of a phosphotyrosine site of the essential STPK PknB reduces its activity in vitro and in live Mtb, indicating that Tyr phosphorylation has a functional role in bacterial growth. These data identify a previously unrecognized phosphorylation system in a human pathogen that claims ∼1.4 million lives every year.
Proceedings of the National Academy of Sciences of the United States of America. 06/2014;
[Show abstract][Hide abstract] ABSTRACT: Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 10(6) bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.
[Show abstract][Hide abstract] ABSTRACT: HIV-1 is taken up by immature monocyte derived dendritic cells (iMDDCs) into tetraspanin rich caves from which the virus can either be transferred to T lymphocytes or enter into endosomes resulting in degradation. HIV-1 binding and fusion with the DC membrane results in low level de novo infection that can also be transferred to T lymphocytes at a later stage. We have previously reported that HIV-1 can induce partial maturation of iMDDCs at both stages of trafficking. Here we show that CD45(+) microvesicles (MV) which contaminate purified HIV-1 inocula due to similar size and density, affect DC maturation, de novo HIV-1 infection and transfer to T lymphocytes. Comparing iMDDCs infected with CD45-depleted HIV-1BaL or matched non-depleted preparations, the presence of CD45(+) MVs was shown to enhance DC maturation and ICAM-1 (CD54) expression, which is involved in DC∶T lymphocyte interactions, while restricting HIV-1 infection of MDDCs. Furthermore, in the DC culture HIV-1 infected (p24(+)) MDDCs were more mature than bystander cells. Depletion of MVs from the HIV-1 inoculum markedly inhibited DC∶T lymphocyte clustering and the induction of alloproliferation as well as limiting HIV-1 transfer from DCs to T lymphocytes. The effects of MV depletion on these functions were reversed by the re-addition of purified MVs from activated but not non-activated SUPT1.CCR5-CL.30 or primary T cells. Analysis of the protein complement of these MVs and of these HIV-1 inocula before and after MV depletion showed that Heat Shock Proteins (HSPs) and nef were the likely DC maturation candidates. Recombinant HSP90α and β and nef all induced DC maturation and ICAM-1 expression, greater when combined. These results suggest that MVs contaminating HIV-1 released from infected T lymphocytes may be biologically important, especially in enhancing T cell activation, during uptake by DCs in vitro and in vivo, particularly as MVs have been detected in the circulation of HIV-1 infected subjects.
[Show abstract][Hide abstract] ABSTRACT: Epithelial-mesenchymal transition (EMT) is a highly conserved morphogenic process defined by the loss of epithelial characteristics and the acquisition of a mesenchymal phenotype. EMT is associated with increased aggressiveness, invasiveness, and metastatic potential in carcinoma cells. To assess the contribution of extracellular vesicles during EMT, we conducted a proteomic analysis of exosomes released from Madin Darby canine kidney (MDCK) cells, and MDCK cells transformed with oncogenic H-Ras (21D1 cells). Exosomes are 40 to 100 nm membranous vesicles originating from the inward budding of late endosomes and multivesicular bodies (MVBs) and are released from cells upon fusion of MVBs with the plasma membrane. Exosomes from MDCK cells (MDCK Exos) and 21D1 cells (21D1Exos) were purified from cell culture media using density gradient centrifugation (OptiPrep), and protein content identified by GeLC MS/MS proteomic profiling. Both MDCK- and 21D1-Exos populations were morphologically similar by cryoelectron microscopy and contained stereotypical exosomes marker proteins such as TSG101, Alix and CD63. In this study we show that the expression levels of typical EMT hallmark proteins seen in whole cells correlate with those observed in MDCKExos and 21D1Exos; i.e., reduction of characteristic inhibitor of angiogenesis, thrombospondin1 and epithelial markers Ecadherin, and EpCAM, with a concomitant up-regulation of mesenchymal makers such as vimentin. Further, we reveal that 21D1Exos are enriched with several proteases (e.g., MMP1, MMP14, MMP19, ADAM10, ADAMTS1), and integrins (e.g., ITGB1, ITGA3, ITGA6) that have been recently implicated in regulating the tumour microenvironment to promote metastatic progression. A salient finding of this study was the unique presence of key transcriptional regulators (e.g., the master transcriptional regulator YXB1) and core splicing complex components (e.g., SF3B1, SF3B3 and SFRS1) in mesenchymal 21D1Exos. Taken together, our findings reveal that exosomes from Ras-transformed MDCK cells are reprogrammed with factors which may be capable of inducing EMT in recipient cells.
[Show abstract][Hide abstract] ABSTRACT: Protein biomarkers have the potential to transform medicine as they are clinically used to diagnose diseases, stratify patients and follow disease states. Even though a large number of potential biomarkers have been proposed over the past few years, almost none of them have been so far implemented in the clinic. One of the reasons for this limited success is the lack of technologies to validate proposed biomarker candidates in larger patient cohorts. This limitation could be alleviated by the use of antibody-independent validation methods such as Selected Reaction Monitoring (SRM). Similar to measurements based on affinity-reagents, SRM based targeted mass spectrometry also requires the generation of definitive assays for each targeted analyte. Here we present a library of SRM assays for 5568 N-glycosites enabling the multiplexed evaluation of clinically relevant N-glycoproteins as biomarker candidates. We demonstrate that this resource can be utilized to select SRM assay sets for cancer-associated N-glycoproteins for their subsequent multiplexed and consistent quantification in 120 human plasma samples. We show that N-glycoproteins spanning five orders of magnitude in abundance can be quantified and that previously reported abundance differences in various cancer types can be recapitulated. Together, the established N-Glycoprotein SRMAtlas resource (available online at http://www.srmatlas.org/) facilitates parallel, efficient, consistent, and sensitive evaluation of proposed biomarker candidates in large clinical sample cohorts.
[Show abstract][Hide abstract] ABSTRACT: Experience from different fields of life sciences suggests that accessible, complete reference maps of the components of the system under study are highly beneficial research tools. Examples of such maps include libraries of the spectroscopic properties of molecules, or databases of drug structures in analytical or forensic chemistry. Such maps, and methods to navigate them, constitute reliable assays to probe any sample for the presence and amount of molecules contained in the map. So far, attempts to generate such maps for any proteome have failed to reach complete proteome coverage. Here we use a strategy based on high-throughput peptide synthesis and mass spectrometry to generate an almost complete reference map (97% of the genome-predicted proteins) of the Saccharomyces cerevisiae proteome. We generated two versions of this mass-spectrometric map, one supporting discovery-driven (shotgun) and the other supporting hypothesis-driven (targeted) proteomic measurements. Together, the two versions of the map constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. To show the utility of the maps, we applied them to a protein quantitative trait locus (QTL) analysis, which requires precise measurement of the same set of peptides over a large number of samples. Protein measurements over 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, influencing the levels of related proteins. Our results suggest that selective pressure favours the acquisition of sets of polymorphisms that adapt protein levels but also maintain the stoichiometry of functionally related pathway members.
[Show abstract][Hide abstract] ABSTRACT: The rigorous testing of hypotheses on suitable sample cohorts is a major limitation in translational research. This is particularly the case for the validation of protein biomarkers; the lack of accurate, reproducible, and sensitive assays for most proteins has precluded the systematic assessment of hundreds of potential marker proteins described in the literature. Here, we describe a high-throughput method for the development and refinement of selected reaction monitoring (SRM) assays for human proteins. The method was applied to generate such assays for more than 1000 cancer-associated proteins, which are functionally related to candidate cancer driver mutations. We used the assays to determine the detectability of the target proteins in two clinically relevant samples: plasma and urine. One hundred eighty-two proteins were detected in depleted plasma, spanning five orders of magnitude in abundance and reaching below a concentration of 10 ng/ml. The narrower concentration range of proteins in urine allowed the detection of 408 proteins. Moreover, we demonstrate that these SRM assays allow reproducible quantification by monitoring 34 biomarker candidates across 83 patient plasma samples. Through public access to the entire assay library, researchers will be able to target their cancer-associated proteins of interest in any sample type using the detectability information in plasma and urine as a guide. The generated expandable reference map of SRM assays for cancer-associated proteins will be a valuable resource for accelerating and planning biomarker verification studies.
Science translational medicine 07/2012; 4(142):142ra94. · 10.76 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Selected reaction monitoring (SRM) is an accurate quantitative technique, typically used for small-molecule mass spectrometry (MS). SRM has emerged as an important technique for targeted and hypothesis-driven proteomic research, and is becoming the reference method for protein quantification in complex biological samples. SRM offers high selectivity, a lower limit of detection and improved reproducibility, compared to conventional shot-gun-based tandem MS (LC-MS/MS) methods. Unlike LC-MS/MS, which requires computationally intensive informatic postanalysis, SRM requires preacquisition bioinformatic analysis to determine proteotypic peptides and optimal transitions to uniquely identify and to accurately quantitate proteins of interest. Extensive arrays of bioinformatics software tools, both web-based and stand-alone, have been published to assist researchers to determine optimal peptides and transition sets. The transitions are oftentimes selected based on preferred precursor charge state, peptide molecular weight, hydrophobicity, fragmentation pattern at a given collision energy (CE), and instrumentation chosen. Validation of the selected transitions for each peptide is critical since peptide performance varies depending on the mass spectrometer used. In this review, we provide an overview of open source and commercial bioinformatic tools for analyzing LC-MS data acquired by SRM.
[Show abstract][Hide abstract] ABSTRACT: Mammalian host response to pathogens is associated with fluctuations in high abundant proteins in body fluids as well as in regulation of proteins expressed in relatively low copy numbers like cytokines secreted from immune cells and endothelium. Hence, efficient monitoring of proteins associated with host response to pathogens remains a challenging task. In this paper, we present a targeted proteome analysis of a panel of 20 proteins that are widely believed to be key players and indicators of bovine host response to mastitis pathogens. Stable isotope-labeled variants of two concordant proteotypic peptides from each of these 20 proteins were obtained through the QconCAT method. We present the quantotypic properties of these 40 proteotypic peptides and discuss their application to research in host-pathogen interactions. Our results clearly demonstrate a robust monitoring of 17 targeted host-response proteins. Twelve of these were readily quantified in a simple extraction of mammary gland tissues, while the expression levels of the remaining proteins were too low for direct and stable quantification; hence, their accurate quantification requires further fractionation of mammary gland tissues.
Journal of Proteome Research 03/2012; 11(3):1832-43. · 5.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Public repositories for proteomics data have accelerated proteomics research by enabling more efficient cross-analyses of datasets, supporting the creation of protein and peptide compendia of experimental results, supporting the development and testing of new software tools, and facilitating the manuscript review process. The repositories available to date have been designed to accommodate either shotgun experiments or generic proteomic data files. Here, we describe a new kind of proteomic data repository for the collection and representation of data from selected reaction monitoring (SRM) measurements. The PeptideAtlas SRM Experiment Library (PASSEL) allows researchers to easily submit proteomic data sets generated by SRM. The raw data are automatically processed in a uniform manner and the results are stored in a database, where they may be downloaded or browsed via a web interface that includes a chromatogram viewer. PASSELenables cross-analysis of SRMdata, supports optimization of SRMdata collection, and facilitates the review process of SRMdata. Further, PASSELwill help in the assessment of proteotypic peptide performance in a wide array of samples containing the same peptide, as well as across multiple experimental protocols.
[Show abstract][Hide abstract] ABSTRACT: Since its inception, proteomics has essentially operated in a discovery mode with the goal of identifying and quantifying the maximal number of proteins in a sample. Increasingly, proteomic measurements are also supporting hypothesis-driven studies, in which a predetermined set of proteins is consistently detected and quantified in multiple samples. Selected reaction monitoring (SRM) is a targeted mass spectrometric technique that supports the detection and quantification of specific proteins in complex samples at high sensitivity and reproducibility. Here, we describe ATAQS, an integrated software platform that supports all stages of targeted, SRM-based proteomics experiments including target selection, transition optimization and post acquisition data analysis. This software will significantly facilitate the use of targeted proteomic techniques and contribute to the generation of highly sensitive, reproducible and complete datasets that are particularly critical for the discovery and validation of targets in hypothesis-driven studies in systems biology.
We introduce a new open source software pipeline, ATAQS (Automated and Targeted Analysis with Quantitative SRM), which consists of a number of modules that collectively support the SRM assay development workflow for targeted proteomic experiments (project management and generation of protein, peptide and transitions and the validation of peptide detection by SRM). ATAQS provides a flexible pipeline for end-users by allowing the workflow to start or end at any point of the pipeline, and for computational biologists, by enabling the easy extension of java algorithm classes for their own algorithm plug-in or connection via an external web site.This integrated system supports all steps in a SRM-based experiment and provides a user-friendly GUI that can be run by any operating system that allows the installation of the Mozilla Firefox web browser.
Targeted proteomics via SRM is a powerful new technique that enables the reproducible and accurate identification and quantification of sets of proteins of interest. ATAQS is the first open-source software that supports all steps of the targeted proteomics workflow. ATAQS also provides software API (Application Program Interface) documentation that enables the addition of new algorithms to each of the workflow steps. The software, installation guide and sample dataset can be found in http://tools.proteomecenter.org/ATAQS/ATAQS.html.
[Show abstract][Hide abstract] ABSTRACT: The Protein Information and Property Explorer 2 (PIPE2) is an enhanced software program and updated web application that aims at providing the proteomic researcher a simple, intuitive user interface through which to begin inquiry into the biological significance of a list of proteins typically produced by MS/MS proteomic processing software. PIPE2 includes an improved interface, new data visualization options, and new data analysis methods for combining disparate, but related, data sets. In particular, PIPE2 has been enhanced to handle multi-dimensional data such as protein abundance, gene expression, and/or interaction data. The current architecture of PIPE2, modeled after that of Gaggle (a programming infrastructure for interoperability between separately developed software tools), contains independent functional units that can be instantiated and pieced together at the user's discretion to form a pipelined analysis workflow. Among these functional units is the Network Viewer component, which adds rich network analysis capabilities to the suite of existing proteomic web resources. Additionally, PIPE2 implements a framework within which new analysis procedures can be easily deployed and distributed over the World Wide Web. PIPE2 is available as a web service at http://pipe2.systemsbiology.net/.