Journal
PROTEOMICS
Volume 8, Issue 3, Pages 435-445Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/pmic.200700680
Keywords
human saliva; peptidomics; protease inhibitors; stable isotope labeling
Funding
- NCI NIH HHS [U24 CA126477] Funding Source: Medline
- NIDCR NIH HHS [U01 DE016274] Funding Source: Medline
- OID CDC HHS [DE-AC02-05CH11231] Funding Source: Medline
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Endogenous proteinases in biological fluids such as human saliva produce a rich peptide repertoire that reflects a unique combination of enzymes, substrates, and inhibitors/activators. Accordingly, this subproteome is an interesting source of biomarkers for disease processes that either directly or indirectly involve proteolysis. However, the relevant proteinases, typically very low abundance molecules, are difficult to classify and identify. We hypothesized that a sensitive technique for monitoring accumulated peptide products in an unbiased, global manner would be very useful for detecting and profiling proteolytic activities in complex biological samples. Building on the longstanding use of O-18 isotope-based approaches for the classification of proteolytic and other enzymatic processes we devised a new method for evaluating endogenous proteinases. Specifically, we showed that upon ex vivo incubation endogenous proteinases in human parotid saliva introduced O-18 from isotopically enriched water into the C-terminal carboxylic groups of their peptide products. Subsequent peptide sequence determination and inhibitor profiling enabled the detection of discrete subsets of proteolytic products that were generated by different enzymes. As a proof-of-principle we used one of these fingerprints to identify the relevant activity as tissue kallikrein. We termed this technique PALeO. Our results suggest that PALeO is a rapid and highly sensitive method for globally assessing proteinase activities in complex biological samples.
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