Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V. , Otto-Hahn-Str. 6b , 44227 Dortmund , Germany.
Gerald Bronfman Department of Oncology , Jewish General Hospital, McGill University , Montreal , Quebec H4A 3T2 , Canada.
J Proteome Res. 2018 May 4;17(5):1923-1933. doi: 10.1021/acs.jproteome.8b00004. Epub 2018 Apr 20.
About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method's versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity.
大约 2%的人类和其他生物体的基因组编码蛋白酶。解析蛋白酶的生物学功能和设计抑制剂的重要步骤是对蛋白酶特异性进行分析。在这项工作中，我们提出了一种新颖的、无需标记的、基于蛋白质组学的蛋白酶特异性分析方法，该方法只需要简单的样品制备步骤。它使用源自蛋白质组的肽文库，并使用移液器吸头中的强阳离子交换色谱 (SCX) 材料对切割序列进行富集。作为该方法通用性的证明，我们成功地确定了 GluC、caspase-3、糜蛋白酶、MMP-1 和组织蛋白酶 G 的特异性，每种蛋白酶的切割事件从几百到近 2000 个不等。有趣的是，我们还发现组织蛋白酶 G 对 P1 亚位点的天冬酰胺有一种新的内在偏好，我们使用合成肽进行了验证。总的来说，该方法简单直接，迄今为止在蛋白酶特异性分析方面所需的材料和设备投资最低。因此，我们认为它将适用于任何生物化学实验室，并促进对蛋白酶特异性的深入了解。
