Biosciences Division, Center for Chemical Biology, SRI International, Harrisonburg, VA, USA.
Proteomics Clin Appl. 2014 Jun;8(5-6):299-307. doi: 10.1002/prca.201300037. Epub 2014 May 11.
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.
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.
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.
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.
蛋白水解酶是很有前途的诊断靶点，因为它们在多种生理过程中发挥着关键作用，并且与许多人类疾病有关。人体血液是一种相对非侵入性的疾病特异性蛋白酶生物标志物检测源，可随后转化为诊断测试。然而，在生物标志物发现的样品制备阶段，选择血清或血浆，更具体地说，选择哪种抗凝剂来制备血浆，是很重要的。
我们之前利用内部荧光猝灭荧光探针组合文库成功绘制了各种生物体液的全局蛋白水解谱。在这项研究中，我们利用该平台确定了三种常用抗凝剂（EDTA、肝素和柠檬酸盐）对从健康白种男性采集的血浆和血清的蛋白水解活性谱的影响。
发现血清和血浆柠檬酸盐的蛋白水解活性最高，其次是血浆肝素，然后是血浆 EDTA。对样品切割和未切割的基序的氨基酸分布进行详细分析，为其中的活性蛋白水解成分提供了重要的见解。
对这些样本的蛋白水解谱进行广泛的定量比较，揭示了与这些生物体液中存在的蛋白酶的差异底物识别相关的几个新的见解。
