Application of Peptide Arrays and Mass Spectrometry to Proteases

描述（由申请人提供）：蛋白水解酶对从细菌到高等真核生物的所有生物体都起着重要作用。了解全基因组测序发现的大量蛋白酶的功能是一个重大挑战，需要新的方法来快速提供蛋白质组范围内的功能信息。在过去的15年中，肽库已被用于确定切割位点序列基序，其可用于设计模型底物，鉴定体内蛋白质底物，并产生有效和特异性的蛋白酶抑制剂。肽微阵列技术的最新进展现在允许肽切割特异性的更高通量分析，但目前的方法只能应用于蛋白酶的子集，并提供有限的信息。我们建议开发一种新的平台，结合肽微阵列与质谱，允许快速，一般和彻底的分析蛋白酶切割的选择性。双标记的荧光共振能量转移肽底物将通过其氨基末端以高密度固定在修饰的载玻片上。用感兴趣的蛋白酶处理载玻片导致肽的子集的位点特异性切割。将在微阵列读取器上检测所产生的荧光变化，从而允许定量评估阵列上每种肽的切割程度。将通过对释放的氨基末端片段进行液相色谱-电喷雾质谱法来确定每个底物肽内的特定切割位点。共有切割基序随后从切割的肽的序列比对衍生。由于蛋白酶广泛参与人类疾病，这种方法有可能影响许多领域的研究和药物发现，包括癌症，关节炎，自身免疫性疾病和传染病。 蛋白酶，分解蛋白质的酶，是正常生理所必需的，但可以在多种情况下导致人类疾病，包括癌症，病毒感染，自身免疫性疾病和阿尔茨海默病。这项研究将开发新的微尺度技术，以快速分析蛋白酶识别其靶蛋白的方式。这些信息将有助于我们了解蛋白酶的功能，并将适用于发现新的蛋白酶抑制剂药物。

DESCRIPTION (provided by applicant): Proteolytic enzymes play essential roles for all living organisms, from bacteria to higher eukaryotes. Understanding the function of the large numbers of proteases that have been uncovered by whole genome sequencing is a significant challenge, and novel methods are needed to rapidly provide functional information on a proteome-wide scale. Peptide libraries have been used over the last 15 years to determine cleavage site sequence motifs, which can be used to design model substrates, identify in vivo protein substrates, and generate potent and specific protease inhibitors. Recent advances in peptide microarray technology now allow higher throughput analysis of peptide cleavage specificity, but current approaches can only be applied to a subset of proteases and provides limited information. We propose to develop a novel platform that combines peptide microarrays with mass spectrometry to allow for rapid, general, and thorough analysis of protease cleavage selectivity. Dually-labeled fluorescence resonance energy transfer peptide substrates will be immobilized on modified glass slides at high density via their amino termini. Treatment of slides with a protease of interest results in site- specific cleavage of a subset of peptides. The resulting change in fluorescence will be detected on a microarray reader, allowing quantitative assessment of the extent of cleavage of each peptide on the array. The specific sites of cleavage within each substrate peptide will be determined by subjecting the released amino terminal fragments to liquid chromatography-electrospray mass spectrometry. Consensus cleavage motifs are subsequently derived from sequence alignments of the cleaved peptides. Because proteases have been implicated widely in human disease, this methodology has the potential to impact research and drug discovery in a number of areas, including cancer, arthritis, autoimmune disease, and infectious disease. Proteases, the enzymes that break down proteins, are essential for normal physiology yet can contribute to human disease in multiple contexts, including cancer, viral infection, autoimmune disease, and Alzheimer's disease. The proposed research will develop new micro-scale technology to rapidly analyze the way that proteases recognize their target proteins. This information will contribute to our understanding of how proteases function and will be applicable to the discovery of new protease inhibitor drugs.