Proteomic characterization of serine hydrolase activity and composition in normal urine

Navarrete et al. Clinical Proteomics 2013, 10:17 http://www.clinicalproteomicsjournal.com/content/10/1/17 CLINICAL PROTEOMICS RESEARCH Open Access Proteomic characterization of serine hydrolase activity and composition in normal urine Mario Navarrete1, Julie Ho1,2*, Oleg Krokhin1, Peyman Ezzati1, Claudio Rigatto3, Martina Reslerova4, David N Rush2, Peter Nickerson1,2,5 and John A Wilkins1 Abstract Background: Serine hydrolases constitute a large enzyme family involved in a diversity of proteolytic and metabolic processes which are essential for many aspects of normal physiology. The roles of serine hydrolases in renal function are largely unknown and monitoring their activity may provide important insights into renal physiology. The goal of this study was to profile urinary serine hydrolases with activity-based protein profiling (ABPP) and to perform an in-depth compositional analysis. Methods: Eighteen healthy individuals provided random, mid-stream urine samples. ABPP was performed by reacting urines (n = 18) with a rhodamine-tagged fluorophosphonate probe and visualizing on SDS-PAGE. Active serine hydrolases were isolated with affinity purification and identified on MS-MS. Enzyme activity was confirmed with substrate specific assays. A complementary 2D LC/MS-MS analysis was performed to evaluate the composition of serine hydrolases in urine. Results: Enzyme activity was closely, but not exclusively, correlated with protein quantity. Affinity purification and MS/MS identified 13 active serine hydrolases. The epithelial sodium channel (ENaC) and calcium channel (TRPV5) regulators, tissue kallikrein and plasmin were identified in active forms, suggesting a potential role in regulating sodium and calcium reabsorption in a healthy human model. Complement C1r subcomponent-like protein, mannan binding lectin serine protease 2 and myeloblastin (proteinase 3) were also identified in active forms. The in-depth compositional analysis identified 62 serine hydrolases in urine independent of activity state. Conclusions: This study identified luminal regulators of electrolyte homeostasis in an active state in the urine, which suggests tissue kallikrein and plasmin may be functionally relevant in healthy individuals. Additional serine hydrolases were identified in an active form that may contribute to regulating innate immunity of the urinary tract. Finally, the optimized ABPP technique in urine demonstrates its feasibility, reproducibility and potential applicability to profiling urinary enzyme activity in different renal physiological and pathophysiological conditions. Keywords: Activity-based protein profiling, Catabolomics, Fluorophosphonate probe, Mass spectrometry Background Detailed knowledge of the composition and activities of urine proteins could provide novel insights into normal renal physiology. Although proteomic studies have identified a large number of urinary proteins [1-4] their functional relevance remains largely unknown. Furthermore, * Correspondence: 1 Manitoba Centre for Proteomics & Systems Biology, 799 John Buhler Research Centre, 715 Mc Dermot Avenue, Winnipeg, Manitoba R3A 1R9, Canada 2 Section of Nephrology, GE 421C Health Sciences Centre, University of Manitoba, 820 Sherbrook Street, Winnipeg, Manitoba R3A 1R9, Canada Full list of author information is available at the end of the article many proteomic studies do not account for post-translational modifications which may have a significant impact on protein function. Many proteins are enzymes that are maintained in a latent state until their activity is required. This allows for rapid host responses, without the time lag required for transcription and translation. Thus, there can be marked changes in functional states in the absence of significant alteration in concentration. These functional changes in activity are undetectable with methods that simply quantify transcript or protein levels, but are important for characterizing the dynamic physiological status of the host. © 2013 Navarrete et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Navarrete et al. Clinical Proteomics 2013, 10:17 http://www.clinicalproteomicsjournal.com/content/10/1/17 Activity-based protein profiling (ABPP) is a novel approach to assess the functional status of selected enzymes in the proteome [5]. ABPP is based on the use of tagged probes that selectively react with the active sites of a given enzyme or family of enzymes [5,6]. Activitybased probes consist of a reactive group that targets the active residue of the enzyme, a short linker and reporter tag. The central premise of ABPP is that accessibility of substrate to the active site of an enzyme is an indicator of enzyme activation. Because the underlying molecular mechanisms of catalysis by members of an enzyme family are often identical [7] it is possible to develop a single probe to detect the active forms of members from a given family [8,9]. Members of the serine hydrolase family share a serine centric charge relay system in their catalytic site and this common feature can be exploited with an activity-based probe to selectively label active serine hydrolases. Furthermore, probe-labeled enzymes can be affinity-purified through their tag and identified by mass spectrometry to determine the specific active enzymes within a biological sample (Figure 1). The serine hydrolase family is one of the largest enzyme classes in humans and constitutes ~1% of predicted protein products from the eukaryotic genome. Serine hydrolases consist of greater than 100 serine proteases and approximately 110 esterases, lipases, peptidases and amidases [10]. While some members are well-characterized (e.g. trypsin, elastase, thrombin, acetylcholinesterase), many have yet to be described [10]. Activity based Probe Page 2 of 11 Indeed the role for ~50% of the non-serine proteases remains undetermined [10], and very little is known about the presence and role of serine hydrolases in the urine of healthy individuals. Therefore the objective of this study was to evaluate the activity and composition of serine hydrolases in normal urine. Thirteen serine hydrolases were identified in an active form in normal urine that may reflect regulation of renal electrolyte homeostasis and innate immunity of the urinary tract. The in-depth compositional analysis identified 62 serine hydrolases in normal urine independent of activity state. The ABPP technique that we optimized in urine is a powerful approach for functional proteomic screening by profiling and identification of enzymes in an active state. Results Demonstration of serine hydrolase activity in normal urine Initial studies were undertaken to determine if there was evidence of serin (...truncated)