A sequence-specific exopeptidase activity test (SSEAT) for "functional" biomarker discovery Journal Article

  


Authors: Villanueva, J.; Nazarian, A.; Lawlor, K.; Yi, S. S.; Robbins, R. J.; Tempst, P.
Article Title: A sequence-specific exopeptidase activity test (SSEAT) for "functional" biomarker discovery
Abstract: One form of functional proteomics entails profiling of genuine activities, as opposed to surrogates of activity or active "states," in a complex biological matrix: for example, tracking enzyme-catalyzed changes, in real time, ranging from imple modifications to complex anabolic or catabolic reactions. Here we present a test to compare defined exoprotease activities within individual proteomes of two or more groups of biological samples. It tracks degradation of artificial substrates, under strictly controlled conditions, using semiautomated MALDI-TOF mass spectrometric analysis of the resulting patterns. Each fragment is quantitated by comparison with double labeled, non-degradable internal standards (all-D-amino acid peptides) spiked into the samples at the same time as the substrates to reflect adsorptive and processing-related losse. The full array of metabolites is then quantitated (coefficients of variation of 6.3-14.3% over five replicates) an subjected to multivariate statistical analysis. Using this approach, we tested serum samples of 48 metastatic thyroid cancer patients and 48 healthy controls, with selected peptide substrates taken from earlier standard peotidomics screens (i.e. the "discovery" phase), and obtained class predictions with 94% sensitivity and 90% specificity without prior feature selection (24 features). The test all but eliminates reproducibility problems related to sample collection, storage, and handling as well as to possible variability in endogenous peptide precursor levels because of hemostatic alterations in cancer patients. © 2008 by The American Society for Biochemistry and Molecular Biology, Inc.
Keywords: controlled study; human cell; case-control studies; sensitivity and specificity; reproducibility; biological marker; reproducibility of results; enzyme degradation; protein depletion; neoplasm proteins; tumor markers, biological; analytic method; enzyme activity; enzyme substrate; automation; proteomics; standard; protein processing; amino acid sequence; molecular sequence data; protein processing, post-translational; kinetics; specimen handling; quantitative analysis; peptides; adsorption; multivariate analysis; thyroid cancer; thyroid neoplasms; metastasis potential; matrix assisted laser desorption ionization time of flight mass spectrometry; metabolite; hemostasis; functional proteomics; spectrometry, mass, matrix-assisted laser desorption-ionization; exopeptidase; covariance; peptidomics; sequence specific exopeptidase activity test; exopeptidases
Journal Title: Molecular & Cellular Proteomics
Volume: 7
Issue: 3
ISSN: 1535-9476
Publisher: Amer Soc Biochemistry Molecular Biology Inc  
Date Published: 2008-03-01
Start Page: 509
End Page: 518
Language: English
DOI: 10.1074/mcp.M700397-MCP200
PUBMED: 17986438
PROVIDER: scopus
DOI/URL:

http://www.scopus.com/inward/record.url?eid=2-s2.0-41549158639&partnerID=40&md5=ff374837f2f56312351a18c9754f1223
Notes: --- - "Cited By (since 1996): 42" - "Export Date: 17 November 2011" - "CODEN: MCPOB" - "Source: Scopus"
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MSK Authors
  1. Jose Villanueva
    24 Villanueva
  2. Paul J Tempst
    324 Tempst
  3. San S Yi
    14 Yi
  4. Kevin S Lawlor
    9 Lawlor
  5. Arpi Gettings
    11 Gettings
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