通过蛋白质N端的选择性酶标记对细胞蛋白水解进行全局分析。
Global analysis of cellular proteolysis by selective enzymatic labeling of protein N-termini.
作者信息
Wiita Arun P, Seaman Julia E, Wells James A
机构信息
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA; Department of Laboratory Medicine, University of California, San Francisco, California, USA.
Department of Pharmaceutical Chemistry, University of California, San Francisco, California, USA.
出版信息
Methods Enzymol. 2014;544:327-58. doi: 10.1016/B978-0-12-417158-9.00013-3.
Proteolysis is a critical modification leading to alteration of protein function with important outcomes in many biological processes. However, for the majority of proteases, we have an incomplete understanding of both cellular substrates and downstream effects. Here, we describe detailed protocols and applications for using the rationally engineered peptide ligase, subtiligase, to specifically label and capture protein N-termini generated by proteases either induced or added to complex biological samples. This method allows identification of the protein targets as well as their precise cleavage locations. This approach has revealed >8000 proteolytic sites in healthy and apoptotic cells including >1700 caspase cleavages. One can further determine substrate preferences through rate analysis with quantitative mass spectrometry, physiological substrate specificities, and even infer the identity of proteases operating in the cell. In this chapter, we also describe how this experimental method can be generalized to investigate proteolysis in any biological sample.
蛋白质水解是一种关键的修饰作用，可导致蛋白质功能改变，在许多生物过程中产生重要结果。然而，对于大多数蛋白酶而言，我们对其细胞底物和下游效应的了解并不完整。在此，我们描述了使用合理设计的肽连接酶枯草杆菌蛋白酶连接酶（subtiligase）的详细方案和应用，以特异性标记和捕获由诱导产生或添加到复杂生物样品中的蛋白酶所生成的蛋白质N端。该方法能够鉴定蛋白质靶点及其精确的切割位点。此方法已揭示了健康细胞和凋亡细胞中超过8000个蛋白水解位点，其中包括超过1700个半胱天冬酶切割位点。通过定量质谱分析的速率分析、生理底物特异性，甚至可以推断细胞中起作用的蛋白酶的身份，从而进一步确定底物偏好。在本章中，我们还描述了如何将这种实验方法推广到研究任何生物样品中的蛋白质水解。
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