A mass spectrometry-based strategy for detecting and characterizing endogenous proteinase activities in complex biological samples.

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.

Proteomic and immunoblot analyses of Bartonella quintana total membrane proteins identify antigens recognized by sera from infected patients.

Bartonella quintana is a fastidious, gram-negative, rod-shaped bacterium that causes prolonged bacteremia in immunocompetent humans and severe infections in immunocompromised individuals. We sought to define the outer membrane subproteome of B. quintana in order to obtain insight into the biology and pathogenesis of this emerging pathogen and to identify the predominant B. quintana antigens targeted by the human immune system during infection. We isolated the total membrane proteins of B. quintana and identified 60 proteins by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mass fingerprinting. Using the newly constructed proteome map, we then utilized two-dimensional immunoblotting with sera from 21 B. quintana-infected patients to identify 24 consistently recognized, immunoreactive B. quintana antigens that have potential relevance for pathogenesis and diagnosis. Among the outer membrane proteins, the variably expressed outer membrane protein adhesins (VompA and VompB), peptidyl-prolyl cis-trans-isomerase (PpI), and hemin-binding protein E (HbpE) were recognized most frequently by sera from patients, which is consistent with surface expression of these virulence factors during human infection.

Assessing the effects of diurnal variation on the composition of human parotid saliva: quantitative analysis of native peptides using iTRAQ reagents.

Changes in salivary composition correlate with disease susceptibility, disease state, or both. However, use of saliva for diagnostic purposes is complicated by the gland-specific effects of circadian rhythm or diurnal variation. We recently characterized a suite of peptides in the < or =10-kDa fraction of human parotid saliva that included many novel species. In this study, we used novel iTRAQ labeling chemistry to investigate possible diurnal effects on peptide generation. We collected samples produced by gustatory stimulation as the ductal secretions at four time points under conditions that minimized proteolysis, pooled them according to collection time, and isolated the LMW fractions. Samples collected at each collection time were derivatized with a different isobaric iTRAQ reagent. The labeled samples were combined, separated by reversed-phase HPLC, co-spotted with matrix on MALDI targets, and analyzed by MALDI TOF/TOF mass spectrometry. With this approach, we achieved relative quantification of the parotid peptides at four time points. In several cases, abundance during the day changed dramatically. iTRAQ tagging improved the efficiency of MS/MS fragmentation, which in turn allowed the identification of several novel peptides. Our results demonstrated both the utility of this method and the importance of diurnal effects on the composition of the human parotid saliva peptidome.

Toward defining the human parotid gland salivary proteome and peptidome: identification and characterization using 2D SDS-PAGE, ultrafiltration, HPLC, and mass spectrometry.

Saliva plays many biological roles, from lubrication and digestion to regulating bacterial and leukocyte adhesion. To understand the functions of individual components and families of molecules, it is important to identify as many salivary proteins as possible. Toward this goal, we used a proteomic approach as the first step in a global analysis of this important body fluid. We collected parotid saliva as the ductal secretion from three human donors and separated the protein components by two-dimensional SDS-polyacrylamide gel electrophoresis (2D SDS-PAGE). Proteins in gel spots were identified by peptide mass fingerprinting, and the results were confirmed by tandem mass spectrometry of selected peptides. Complementing this approach we used ultrafiltration to prepare a low-molecular-weight fraction of parotid saliva, which was analyzed directly or after reversed phase high-performance liquid chromatography separation by using mass spectrometric approaches. MS analyses of 2D SDS-PAGE spots revealed known components of saliva, including cystatins, histatins, lysozyme, and isoforms and/or fragments of alpha-amylase, albumin, and proline-rich proteins. We also discovered novel proteins, such as several isoforms of Zn-alpha-2-glycoprotein and secretory actin-binding protein. MS analyses of the ultrafiltrate showed that the low-molecular-weight fraction of parotid saliva was peptide-rich, with novel fragments of proline-rich proteins and histatins in abundance. Experiments using Candida albicans as the test organism showed that at least one of the novel peptides had antifungal activity. Our results show that saliva is a rich source of proteins and peptides that are potential diagnostic and therapeutic targets.