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dc.contributor.author Navarrete, Mario
dc.contributor.author Ho, Julie
dc.contributor.author Krokhin, Oleg
dc.contributor.author Ezzati, Peyman
dc.contributor.author Rigatto, Claudio
dc.contributor.author Reslerova, Martina
dc.contributor.author Rush, David N
dc.contributor.author Nickerson, Peter
dc.contributor.author Wilkins, John A
dc.date.accessioned 2014-04-02T10:48:19Z
dc.date.available 2014-04-02T10:48:19Z
dc.date.issued 2013-11-15
dc.identifier.citation Clinical Proteomics. 2013 Nov 15;10(1):17
dc.identifier.uri http://hdl.handle.net/1993/23360
dc.description.abstract 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.
dc.title Proteomic characterization of serine hydrolase activity and composition in normal urine
dc.type Journal Article
dc.language.rfc3066 en
dc.description.version Peer Reviewed
dc.rights.holder Mario Navarrete et al.; licensee BioMed Central Ltd.
dc.date.updated 2014-04-02T10:48:20Z
dc.identifier.doi http://dx.doi.org/10.1186/1559-0275-10-17


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