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首页> 外文期刊>Mass Spectrometry Reviews >GETTING INTIMATE WITH TRYPSIN, THE LEADING PROTEASE IN PROTEOMICS
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GETTING INTIMATE WITH TRYPSIN, THE LEADING PROTEASE IN PROTEOMICS

机译：与胰蛋白酶（蛋白质组学中的主要蛋白酶）亲密接触

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Nowadays, mass spectrometry-based proteomics is carried out primarily in a bottom-up fashion, with peptides obtained after proteolytic digest of a whole proteome lysate as the primary analytes instead of the proteins themselves. This experimental setup crucially relies on a protease to digest an abundant and complex protein mixture into a far more complex peptide mixture. Full knowledge of the working mechanism and specificity of the used proteases is therefore crucial, both for the digestion step itself as well as for the downstream identification and quantification of the (fragmentation) mass spectra acquired for the peptides in the mixture. Targeted protein analysis through selected reaction monitoring, a relative newcomer in the specific field of mass spectrometry-based proteomics, even requires a priori understanding of protease behavior for the proteins of interest. Because of the rapidly increasing popularity of proteomics as an analytical tool in the life sciences, there is now a renewed demand for detailed knowledge on trypsin, the workhorse protease in proteomics. This review addresses this need and provides an overview on the structure and working mechanism of trypsin, followed by a critical analysis of its cleavage behavior, typically simply accepted to occur exclusively yet consistently after Arg andLys, unless they are followed by a Pro. In this context, shortcomings in our ability to understand and predict the behavior of trypsin will be highlighted, along with the downstream implications. Furthermore, an analysis is carried out on the inherent shortcomings of trypsin with regard to whole proteome analysis, and alternative approaches will be presented that can alleviate these issues. Finally, some reflections on the future of trypsin as the workhorse protease in mass spectrometry-based proteomics will be provided. ? 2013 Wiley Periodicals, Inc. Mass Spec Rev 32:453-465,2013
机译：如今，基于质谱的蛋白质组学主要以自下而上的方式进行，蛋白质水解后是将整个蛋白质组裂解物进行蛋白水解消化后得到的肽作为主要分析物，而不是蛋白质本身。该实验装置至关重要地依赖蛋白酶将大量复杂的蛋白质混合物消化成更复杂的肽混合物。因此，对于消化步骤本身以及对于混合物中的肽所获得的（片段化）质谱的下游鉴定和定量而言，充分了解所用蛋白酶的工作机理和特异性至关重要。通过选定的反应监测进行靶向蛋白质分析，这是基于质谱的蛋白质组学特定领域中的一个相对较新的事物，甚至需要事先了解目标蛋白质的蛋白酶行为。由于蛋白质组学作为生命科学中的一种分析工具迅速普及，现在对胰蛋白酶（蛋白质组学中的主力蛋白酶）的详细知识有了新的需求。这篇综述解决了这一需求，并概述了胰蛋白酶的结构和工作机制，然后对其裂解行为进行了批判性分析，通常除非经过Pro和专家的认可，否则通常仅被接受仅在Arg和Lys之后一致发生。在这种情况下，我们在理解和预测胰蛋白酶行为方面的能力缺陷以及下游影响将被突出。此外，针对整个蛋白质组分析，对胰蛋白酶的固有缺点进行了分析，并将提出可减轻这些问题的替代方法。最后，将提供有关胰蛋白酶作为基于质谱的蛋白质组学中的主力蛋白酶的未来的一些思考。 ？ 2013 Wiley Periodicals，Inc.质谱Rev 32：453-465,2013

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