Sampling the N-terminal proteome of human blood

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/2010; 107(10):4561-6. DOI: 10.1073/pnas.0914495107
Source: PubMed


The proteomes of blood plasma and serum represent a potential gold mine of biological and diagnostic information, but challenges such as dynamic range of protein concentration have hampered efforts to unlock this resource. Here we present a method to label and isolate N-terminal peptides from human plasma and serum. This process dramatically reduces the complexity of the sample by eliminating internal peptides. We identify 772 unique N-terminal peptides in 222 proteins, ranging over six orders of magnitude in abundance. This approach is highly suited for studying natural proteolysis in plasma and serum. We find internal cleavages in plasma proteins created by endo- and exopeptidases, providing information about the activities of proteolytic enzymes in blood, which may be correlated with disease states. We also find signatures of signal peptide cleavage, coagulation and complement activation, and other known proteolytic processes, in addition to a large number of cleavages that have not been reported previously, including over 200 cleavages of blood proteins by aminopeptidases. Finally, we can identify substrates from specific proteases by exogenous addition of the protease combined with N-terminal isolation and quantitative mass spectrometry. In this way we identified proteins cleaved in human plasma by membrane-type serine protease 1, an enzyme linked to cancer progression. These studies demonstrate the utility of direct N-terminal labeling by subtiligase to identify and characterize endogenous and exogenous proteolysis in human plasma and serum.

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    • "advantage that all naturally modified protein N-or C-termini are retained, enabling profiling of known and discovery of new terminal modifications, irrespective of their chemical identity (Fig. 2A). Although most published terminomics studies focus on protease substrate identification in relatively simple in vitro or cell-based assays, recent studies demonstrated the utility of N-terminomics approaches for characterization of protein termini in complex tissues, including inflamed skin [7], platelets [54], plasma [55], human erythrocytes [33], and human placenta (own unpublished data). Streamlined terminomics workflows have recently been established for proteome-wide mapping of protein termini as part of the human proteome project [33] [56]. "
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    ABSTRACT: Early accurate diagnosis and personalized treatment are essential in order to treat complex or fatal diseases such as cancer and autoimmune, cardiovascular and neurodegenerative diseases. To realize this vision, new diagnostic and prognostic biomarkers are urgently required. MS-based proteomics is the most promising approach for protein biomarker identification, but suffers in clinical translation of biomarker candidates that show only quantitative differences from normal tissue. Indeed, success in translating proteomic data to biomarkers in the clinic has been disappointing. Here, we propose that protein termini provide a new opportunity for biomarker discovery due to qualitative differences in intact and new protein termini between diseased and normal tissues. Altered proteolysis occurs in most pathologies. Disease- and process-specific protein modifications, including proteolytic processing and subsequent modification of the terminal amino acids, frequently lead to altered protein activity that plays key roles in the disease process. Thus, mapping of ensembles of characteristic protein termini provides a proteolytic signature of high information content that shows both quantitative and most importantly qualitative differences in different diseases and stage of disease. These unique protein biomarkers have the added benefit of being mechanistically informative by revealing the activity state of the bioactive protein. Moreover, proteome-wide isolation of protein termini leads to generalized sample simplification, thereby enabling up to three orders of magnitude lower LODs compared to traditional shotgun proteomic approaches. We introduce the potential of protein termini for biomarker discovery, briefly review methods enabling large-scale studies of protein termini, and discuss how these may be integrated into a termini-oriented biomarker discovery pipeline from discovery to clinical application.
    Full-text · Article · Jun 2014 · PROTEOMICS - CLINICAL APPLICATIONS
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    • "Nevertheless, the study did not identify any notable differences in mean analyte levels between EDTA plasma and P100 plasma, despite the presence of protease inhibitors in the P100 blood collection tubes. These results are similar to the findings of others using mass spectroscopy and multiplex ELISA methods [18-20]. Enzymatic degradation has been reported to occur during blood collection and processing [5,21]; protease inhibitors have stabilized the proteome in some studies [22]. "
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    Full-text · Article · Jan 2014 · Journal of Translational Medicine
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    • "UniProt gene identifiers for homologous human genes were obtained for each unique, differentially expressed black bear protein (n = 15) that was identified by MS/MS. A list of the N-terminal serum proteome of human blood (n = 213) was used as the background total gene list [27] because similar data does not exist for the American black bear. The enrichment of each GO category was calculated as the proportion of changed to total proteins for each category. "
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    Full-text · Article · Jun 2013 · PLoS ONE
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