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

Josep Villanueva, Arpi Nazarian, Kevin Lawlor, San San Yi, Richard J. Robbins, Paul Tempst

Research output: Contribution to journalArticlepeer-review

83 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)509-518
Number of pages10
JournalMolecular and Cellular Proteomics
Issue number3
StatePublished - Mar 2008

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Molecular Biology


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