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Open Access Highly Accessed Research

Proteomic characterization of serine hydrolase activity and composition in normal urine

Mario Navarrete1, Julie Ho12*, Oleg Krokhin1, Peyman Ezzati1, Claudio Rigatto3, Martina Reslerova4, David N Rush2, Peter Nickerson125 and John A Wilkins1

Author Affiliations

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

3 Section of Nephrology, Renal Health Program - Seven Oaks General Hospital/SBGH, University of Manitoba, 2PD02-2300 McPhillips Street, Winnipeg, Manitoba R2V 3M3, Canada

4 Section of Nephrology, St. Boniface General Hospital, University of Manitoba, 409 Tache Avenue, Winnipeg, Manitoba R2H 2A6, Canada

5 Organ & Tissues Office, Canadian Blood Services Building, Room 312-777 William Avenue, Winnipeg, Manitoba R3E 3P4, Canada

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Clinical Proteomics 2013, 10:17  doi:10.1186/1559-0275-10-17

Published: 15 November 2013

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 =‚ÄČ18) 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