Kalyani Jambunathan1, Amit K Galande. 1. Biosciences Division, Center for Chemical Biology, SRI International, Harrisonburg, VA, USA.
Abstract
PURPOSE: Proteolytic enzymes are promising diagnostic targets since they play key roles in diverse physiological processes and have been implicated in numerous human diseases. Human blood is a relatively noninvasive source for disease-specific protease biomarker detection and subsequent translation into diagnostic tests. However, the choice of serum or plasma, and more specifically, which anticoagulant to choose in plasma preparation, is important to address in the sample preparation phase of biomarker discovery. EXPERIMENTAL DESIGN: We have previously utilized a combinatorial library of internally quenched fluorogenic probes to successfully map the global proteolytic profiles of various biological fluids. In this study, we utilized the platform to ascertain the impact of three commonly used anticoagulants (EDTA, heparin, and citrate) on the proteolytic activity profile of plasma and serum collected from a healthy Caucasian male. RESULTS: Serum and plasma citrate were observed to be most proteolytically active, followed by plasma heparin and then plasma EDTA. Detailed analysis of the amino acid distribution of motifs cleaved and not cleaved by the samples offered significant insights in to active proteolytic components within them. CONCLUSION AND CLINICAL RELEVANCE: Broad quantitative comparison of proteolytic profiles of these samples revealed several novel insights related to the differential substrate recognition of proteases present in these biological fluids.
PURPOSE: Proteolytic enzymes are promising diagnostic targets since they play key roles in diverse physiological processes and have been implicated in numerous human diseases. Human blood is a relatively noninvasive source for disease-specific protease biomarker detection and subsequent translation into diagnostic tests. However, the choice of serum or plasma, and more specifically, which anticoagulant to choose in plasma preparation, is important to address in the sample preparation phase of biomarker discovery. EXPERIMENTAL DESIGN: We have previously utilized a combinatorial library of internally quenched fluorogenic probes to successfully map the global proteolytic profiles of various biological fluids. In this study, we utilized the platform to ascertain the impact of three commonly used anticoagulants (EDTA, heparin, and citrate) on the proteolytic activity profile of plasma and serum collected from a healthy Caucasian male. RESULTS: Serum and plasma citrate were observed to be most proteolytically active, followed by plasma heparin and then plasma EDTA. Detailed analysis of the amino acid distribution of motifs cleaved and not cleaved by the samples offered significant insights in to active proteolytic components within them. CONCLUSION AND CLINICAL RELEVANCE: Broad quantitative comparison of proteolytic profiles of these samples revealed several novel insights related to the differential substrate recognition of proteases present in these biological fluids.
Authors: Quentin M Nunes; Dunhao Su; Philip J Brownridge; Deborah M Simpson; Changye Sun; Yong Li; Thao P Bui; Xiaoying Zhang; Wei Huang; Daniel J Rigden; Robert J Beynon; Robert Sutton; David G Fernig Journal: PLoS One Date: 2019-06-18 Impact factor: 3.240
Authors: Vinh A Nguyen; Leeanne M Carey; Loretta Giummarra; Pierre Faou; Ira Cooke; David W Howells; Tamara Tse; S Lance Macaulay; Henry Ma; Stephen M Davis; Geoffrey A Donnan; Sheila G Crewther Journal: Front Neurol Date: 2016-06-14 Impact factor: 4.003