title: A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samples.
abstract: 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 18O 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 18O 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.
authors.affiliation: Department of Cell and Tissue Biology, University of California-San Francisco, San Francisco, CA, USA.
authors.forename: Sarah
authors.initials: S
authors.lastname: Robinson
authors.forename: Richard K
authors.initials: RK
authors.lastname: Niles
authors.forename: H Ewa
authors.initials: HE
authors.lastname: Witkowska
authors.forename: Kirsten J
authors.initials: KJ
authors.lastname: Rittenbach
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Nichols
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Sargent
authors.forename: Scott E
authors.initials: SE
authors.lastname: Dixon
authors.forename: Akraporn
authors.initials: A
authors.lastname: Prakobphol
authors.forename: Steven C
authors.initials: SC
authors.lastname: Hall
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Fisher
authors.forename: Markus
authors.initials: M
authors.lastname: Hardt
mesh_terms: D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000032:analysis*; D006801:Humans; D008969:Molecular Sequence Data; D010103:Oxygen Isotopes / Q000737:chemistry; D010306:Parotid Gland / Q000378:metabolism; D010455:Peptides / Q000032:analysis*; D020543:Proteome / Q000378:metabolism*; D012463:Saliva / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014867:Water / Q000737:chemistry
chemical_list: D010103:Oxygen Isotopes; D010455:Peptides; D020543:Proteome; D014867:Water; D010450:Endopeptidases