Molecular & Cellular Proteomics

Journal: Molecular & Cellular Proteomics

Abbreviation

Mol Cell Proteomics

Publisher

American Society for Biochemistry and Molecular Biology

ISSN

1535-9476
1535-9484

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Publications1 - 10 of 130

A High-Confidence Human Plasma Proteome Reference Set with Estimated Concentrations in PeptideAtlas
Farrah, Terry; Deutsch, Eric W.; Omenn, Gilbert S.; et al. (2011)
Molecular & Cellular Proteomics
Human blood plasma can be obtained relatively noninvasively and contains proteins from most, if not all, tissues of the body. Therefore, an extensive, quantitative catalog of plasma proteins is an important starting point for the discovery of disease biomarkers. In 2005, we showed that different proteomics measurements using different sample preparation and analysis techniques identify significantly different sets of proteins, and that a comprehensive plasma proteome can be compiled only by combining data from many different experiments. Applying advanced computational methods developed for the analysis and integration of very large and diverse data sets generated by tandem MS measurements of tryptic peptides, we have now compiled a high-confidence human plasma proteome reference set with well over twice the identified proteins of previous high-confidence sets. It includes a hierarchy of protein identifications at different levels of redundancy following a clearly defined scheme, which we propose as a standard that can be applied to any proteomics data set to facilitate cross-proteome analyses. Further, to aid in development of blood-based diagnostics using techniques such as selected reaction monitoring, we provide a rough estimate of protein concentrations using spectral counting. We identified 20,433 distinct peptides, from which we inferred a highly nonredundant set of 1929 protein sequences at a false discovery rate of 1%. We have made this resource available via PeptideAtlas, a large, multiorganism, publicly accessible compendium of peptides identified in tandem MS experiments conductedby laboratories around the world.
The Mouse C2C12 Myoblast Cell Surface N-Linked Glycoproteome
Gundry, Rebekah L.; Raginski, Kimberly; Tarasova, Yelena; et al. (2009)
Molecular & Cellular Proteomics
Endogenous regeneration and repair mechanisms are responsible for replacing dead and damaged cells to maintain or enhance tissue and organ function, and one of the best examples of endogenous repair mechanisms involves skeletal muscle. Although the molecular mechanisms that regulate the differentiation of satellite cells and myoblasts toward myofibers are not fully understood, cell surface proteins that sense and respond to their environment play an important role. The cell surface capturing technology was used here to uncover the cell surface N-linked glycoprotein subproteome of myoblasts and to identify potential markers of myoblast differentiation. 128 bona fide cell surface-exposed N-linked glycoproteins, including 117 transmembrane, four glycosylphosphatidylinositol-anchored, five extracellular matrix, and two membrane-associated proteins were identified from mouse C2C12 myoblasts. The data set revealed 36 cluster of differentiation-annotated proteins and confirmed the occupancy for 235 N-linked glycosylation sites. The identification of the N-glycosylation sites on the extracellular domain of the proteins allowed for the determination of the orientation of the identified proteins within the plasma membrane. One glycoprotein transmembrane orientation was found to be inconsistent with Swiss-Prot annotations, whereas ambiguous annotations for 14 other proteins were resolved. Several of the identified N-linked glycoproteins, including aquaporin-1 and β-sarcoglycan, were found in validation experiments to change in overall abundance as the myoblasts differentiate toward myotubes. Therefore, the strategy and data presented shed new light on the complexity of the myoblast cell surface subproteome and reveal new targets for the clinically important characterization of cell intermediates during myoblast differentiation into myotubes.
Extending the Limits of Quantitative Proteome Profiling with Data-Independent Acquisition and Application to Acetaminophen-Treated Three-Dimensional Liver Microtissues
Bruderer, Roland; Bernhardt, Oliver M.; Gandhi, Tejas; et al. (2015)
Molecular & Cellular Proteomics
The data-independent acquisition (DIA) approach has recently been introduced as a novel mass spectrometric method that promises to combine the high content aspect of shotgun proteomics with the reproducibility and precision of selected reaction monitoring. Here, we evaluate, whether SWATH-MS type DIA effectively translates into a better protein profiling as compared with the established shotgun proteomics. We implemented a novel DIA method on the widely used Orbitrap platform and used retention-time-normalized (iRT) spectral libraries for targeted data extraction using Spectronaut. We call this combination hyper reaction monitoring (HRM). Using a controlled sample set, we show that HRM outperformed shotgun proteomics both in the number of consistently identified peptides across multiple measurements and quantification of differentially abundant proteins. The reproducibility of HRM in peptide detection was above 98%, resulting in quasi complete data sets compared with 49% of shotgun proteomics. Utilizing HRM, we profiled acetaminophen (APAP)1 treated three-dimensional human liver microtissues. An early onset of relevant proteome changes was revealed at subtoxic doses of APAP. Further, we detected and quantified for the first time human NAPQI-protein adducts that might be relevant for the toxicity of APAP. The adducts were identified on four mitochondrial oxidative stress related proteins (GATM, PARK7, PRDX6, and VDAC2) and two other proteins (ANXA2 and FTCD). Our findings imply that DIA should be the preferred method for quantitative protein profiling.
qcML: An Exchange Format for Quality Control Metrics from Mass Spectrometry Experiments
Walzer, Mathias; Espona Pernas, Lucia; Nasso, Sara; et al. (2014)
Molecular & Cellular Proteomics
Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data. What has been missing, however, is a standard data exchange format for reporting these performance metrics. We therefore developed the qcML format, an XML-based standard that follows the design principles of the related mzML, mzIdentML, mzQuantML, and TraML standards from the HUPO-PSI (Proteomics Standards Initiative). In addition to the XML format, we also provide tools for the calculation of a wide range of quality metrics as well as a database format and interconversion tools, so that existing LIMS systems can easily add relational storage of the quality control data to their existing schema. We here describe the qcML specification, along with possible use cases and an illustrative example of the subsequent analysis possibilities. All information about qcML is available at http://code.google.com/p/qcml.
Multiplex N-terminome Analysis of MMP-2 and MMP-9 Substrate Degradomes by iTRAQ-TAILS Quantitative Proteomics
Prudova, Anna; auf dem Keller, Ulrich; Butler, Georgina S.; et al. (2010)
Molecular & Cellular Proteomics
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 using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.
Mass Spectrometric Detection of Tissue Proteins in Plasma
Zhang, Hui; Liu, Alvin Y.; Loriaux, Paul; et al. (2007)
Molecular & Cellular Proteomics
It has long been thought that blood plasma could serve as a window into the state of one’s organs in health and disease because tissue-derived proteins represent a significant fraction of the plasma proteome. Although substantial technical progress has been made toward the goal of comprehensively analyzing the blood plasma proteome, the basic assumption that proteins derived from a variety of tissues could indeed be detectable in plasma using current proteomics technologies has not been rigorously tested. Here we provide evidence that such tissue-derived proteins are both present and detectable in plasma via direct mass spectrometric analysis of captured glycopeptides and thus provide a conceptual basis for plasma protein biomarker discovery and analysis.
Systems-level Proteomics of Two Ubiquitous Leaf Commensals Reveals Complementary Adaptive Traits for Phyllosphere Colonization
Müller, Daniel B.; Schubert, Olga T.; Rost, Hannes; et al. (2016)
Molecular & Cellular Proteomics
Plants are colonized by a diverse community of microorganisms, the plant microbiota, exhibiting a defined and conserved taxonomic structure. Niche separation based on spatial segregation and complementary adaptation strategies likely forms the basis for coexistence of the various microorganisms in the plant environment. To gain insights into organism-specific adaptations on a molecular level, we selected two exemplary community members of the core leaf microbiota and profiled their proteomes upon Arabidopsis phyllosphere colonization. The highly quantitative mass spectrometric technique SWATH MS was used and allowed for the analysis of over two thousand proteins spanning more than three orders of magnitude in abundance for each of the model strains. The data suggest that Sphingomonas melonis utilizes amino acids and hydrocarbon compounds during colonization of leaves whereas Methylobacterium extorquens relies on methanol metabolism in addition to oxalate metabolism, aerobic anoxygenic photosynthesis and alkanesulfonate utilization. Comparative genomic analyses indicates that utilization of oxalate and alkanesulfonates is widespread among leaf microbiota members whereas, aerobic anoxygenic photosynthesis is almost exclusively found in Methylobacteria. Despite the apparent niche separation between these two strains we also found a relatively small subset of proteins to be coregulated, indicating common mechanisms, underlying successful leaf colonization. Overall, our results reveal for two ubiquitous phyllosphere commensals species-specific adaptations to the host environment and provide evidence for niche separation within the plant microbiota.
Protein identification
Bradshaw, Ralph A.; Burlingame, Alma L.; Carr, Steve; et al. (2005)
Molecular & Cellular Proteomics
In Vivo Assessment of Protease Dynamics in Cutaneous Wound Healing by Degradomics Analysis of Porcine Wound Exudates
Sabino, Fabio; Hermes, Olivia; Egli, Fabian; et al. (2015)
Molecular & Cellular Proteomics
Proteases control complex tissue responses by modulating inflammation, cell proliferation and migration, and matrix remodeling. All these processes are orchestrated in cutaneous wound healing to restore the skin's barrier function upon injury. Altered protease activity has been implicated in the pathogenesis of healing impairments, and proteases are important targets in diagnosis and therapy of this pathology. Global assessment of proteolysis at critical turning points after injury will define crucial events in acute healing that might be disturbed in healing disorders. As optimal biospecimens, wound exudates contain an ideal proteome to detect extracellular proteolytic events, are noninvasively accessible, and can be collected at multiple time points along the healing process from the same wound in the clinics. In this study, we applied multiplexed Terminal Amine Isotopic Labeling of Substrates (TAILS) to globally assess proteolysis in early phases of cutaneous wound healing. By quantitative analysis of proteins and protein N termini in wound fluids from a clinically relevant pig wound model, we identified more than 650 proteins and discerned major healing phases through distinctive abundance clustering of markers of inflammation, granulation tissue formation, and re-epithelialization. TAILS revealed a high degree of proteolysis at all time points after injury by detecting almost 1300 N-terminal peptides in ∼450 proteins. Quantitative positional proteomics mapped pivotal interdependent processing events in the blood coagulation and complement cascades, temporally discerned clotting and fibrinolysis during the healing process, and detected processing of complement C3 at distinct time points after wounding and by different proteases. Exploiting data on primary cleavage specificities, we related candidate proteases to cleavage events and revealed processing of the integrin adapter protein kindlin-3 by caspase-3, generating new hypotheses for protease-substrate relations in the healing skin wound in vivo. The data have been deposited to the ProteomeXchange Consortium with identifier PXD001198.
Proteome Analysis of Halobacterium sp. NRC-1 Facilitated by the Biomodule Analysis Tool BMSorter
Gan, Rueichi R.; Yi, Eugene C.; Chiu, Yulun; et al. (2006)
Molecular & Cellular Proteomics
To better understand the extremely halophilic archaeon Halobacterium species NRC-1, we analyzed its soluble proteome by two-dimensional liquid chromatography coupled to electrospray ionization tandem mass spectrometry. A total of 888 unique proteins were identified with a ProteinProphet probability (P) between 0.9 and 1.0. To evaluate the biochemical activities of the organism, the proteomic data were subjected to a biological network analysis using our BMSorter software. This allowed us to examine the proteins expressed in different biomodules and study the interactions between pertinent biomodules. Interestingly an integrated analysis of the enzymes in the amino acid metabolism and citrate cycle networks suggested that up to eight amino acids may be converted to oxaloacetate, fumarate, or oxoglutarate in the citrate cycle for energy production. In addition, glutamate and aspartate may be interconverted from other amino acids or synthesized from citrate cycle intermediates to meet the high demand for the acidic amino acids that are required to build the highly acidic proteome of the organism. Thus this study demonstrated that proteome analysis can provide useful information and help systems analyses of organisms.

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