Protease Specificity Profiling in a Pipet Tip Using “Charge-Synchronized” Proteome-Derived Peptide Libraries

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.人类及其他生物的基因组中，约有2%的序列编码蛋白酶（protease）。解析蛋白酶的生物学功能并设计其抑制剂的核心步骤之一，是开展蛋白酶特异性谱分析。本研究提出了一种基于蛋白质组学的新型无标记蛋白酶特异性谱分析方法，仅需简易的样品制备流程。该方法采用蛋白质组衍生的肽库，并通过移液枪头中的强阳离子交换色谱（strong cation exchange chromatography, SCX）材料富集酶切后的肽段序列。为验证该方法的通用性，我们成功解析了GluC、半胱天冬酶-3（caspase-3）、胰凝乳蛋白酶、基质金属蛋白酶-1（MMP-1）以及组织蛋白酶G的特异性，每个蛋白酶可获取数百至近2000个酶切事件数据。值得注意的是，我们还发现组织蛋白酶G对P1亚位点的天冬酰胺（Asn）具有全新的内在偏好性，并通过合成肽段验证了这一结果。总体而言，该方法操作简便，是目前已知的蛋白酶特异性谱分析中耗材与设备投入最低的技术方案。因此，我们认为该方法可适配任意生物化学实验室，并将推动学界对蛋白酶特异性的认知进一步深化。