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.

中文翻译：

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使用“电荷同步”蛋白质组衍生肽库在移液枪头中进行蛋白酶特异性分析

人类和其他生物的基因组中约2％编码蛋白酶。破解蛋白酶的生物学功能和设计抑制剂的重要步骤是蛋白酶特异性的概况分析。在这项工作中，我们提出了一种新颖的，无标记的，基于蛋白质组学的蛋白酶特异性谱分析方法，该方法仅需要简单的样品制备步骤。它使用蛋白质组衍生的肽库，并在移液器吸头中使用强阳离子交换色谱（SCX）材料富集裂解序列。为了证明该方法的多功能性，我们成功地从每种蛋白酶的数百个至几乎2000个裂解事件中确定了GluC，caspase-3，糜蛋白酶，MMP-1和组织蛋白酶G的特异性。有趣的是，我们还在P1亚位点发现了组织蛋白酶G对Asn的新的内在偏好，我们使用合成肽证实了这一点。总的来说，该方法简单易行，迄今为止，其在蛋白酶特异性分析方面的材料和设备投资最少。因此，我们认为它将适用于任何生物化学实验室，并促进人们对蛋白酶特异性的进一步了解。