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.

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

“…Tryptic and native peptides in these fractions were analyzed by either LC-MALDI TOF/TOF MS (4800, Applied Biosystems, Foster City, CA) or LC-QqTOF MS on an Applied Biosystems QSTAR Pulsar XL instrument equipped with a nanoelectrospray interface. 37 MS data were analyzed using a laboratory information system, created in-house, that uses Mascot Distiller for spectral processing and peak detection. Peptide identifications were accomplished using the Mascot algorithm (version 2.1) to search against human proteins in the Swiss-Prot (version 50; release date May 2, 2006) and IPI (version 3.15; release date February 22, 2006) databases.…”

Agonist-biased Trafficking of Somatostatin Receptor 2A in Enteric Neurons

“…Samples were analyzed using a MALDI-TOF/TOF mass spectrometer (4800 proteomics analyzer, Applied Biosystems, Bedford, MA). Peptide identities were confirmed by MS/MS sequencing, and 18 O-incorporation ratios were determined as described previously (12).…”

“…To define the kinetics and sequence of SST-14 and SST-28 hydrolysis, we monitored the appearance of products using the activity-based mass spectrometry proteinase activity labeling employing 18 O-enriched water (12). Newly formed cleavage products were identified by the incorporation of 18 O atoms into nascent C-terminal carboxylic groups, allowing for precise determination of the sequence of degradation.…”

“…Products were separated by high-pressure liquid chromatography (3). To determine kinetics of hydrolysis at specific sites, we used proteinase activity labeling employing 18 O-enriched water (12,13). SST-14 or SST-28 (100 M) were incubated with ECE-1 (100 nM) in buffers containing 1:1 (v/v) H 2 18 O (Sigma-Aldrich, St. Louis, MO) (0 -48 h, room temperature).…”

Endosomal Endothelin-converting Enzyme-1

“…Samples were prepared and analyzed in triplicate using a 4800 Proteomics Analyzer MALDI-TOF mass spectrometer (Applied Biosystems, Foster City, CA). Peptides were identified, and 18 O incorporation ratios were determined (23).…”