State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences • Beijing, Beijing Institute of Lifeomics, Beijing, China.
Innovation Institute of Medical School, Medical College, Qingdao University, Qingdao, China.
Nat Protoc. 2020 Sep;15(9):2891-2919. doi: 10.1038/s41596-020-0352-2. Epub 2020 Jul 20.
Cysteine is unique among all protein-coding amino acids, owing to its intrinsically high nucleophilicity. The cysteinyl thiol group can be covalently modified by a broad range of redox mechanisms or by various electrophiles derived from exogenous or endogenous sources. Measuring the response of protein cysteines to redox perturbation or electrophiles is critical for understanding the underlying mechanisms involved. Activity-based protein profiling based on thiol-reactive probes has been the method of choice for such analyses. We therefore adapted this approach and developed a new chemoproteomic platform, termed 'QTRP' (quantitative thiol reactivity profiling), that relies on the ability of a commercially available thiol-reactive probe IPM (2-iodo-N-(prop-2-yn-1-yl)acetamide) to covalently label, enrich and quantify the reactive cysteinome in cells and tissues. Here, we provide a detailed and updated workflow of QTRP that includes procedures for (i) labeling of the reactive cysteinome from cell or tissue samples (e.g., control versus treatment) with IPM, (ii) processing the protein samples into tryptic peptides and tagging the probe-modified peptides with isotopically labeled azido-biotin reagents containing a photo-cleavable linker via click chemistry reaction, (iii) capturing biotin-conjugated peptides with streptavidin beads, (iv) identifying and quantifying the photo-released peptides by mass spectrometry (MS)-based shotgun proteomics and (v) interpreting MS data by a streamlined informatic pipeline using a proteomics software, pFind 3, and an automatic post-processing algorithm. We also exemplified here how to use QTRP for mining HO-sensitive cysteines and for determining the intrinsic reactivity of cysteines in a complex proteome. We anticipate that this protocol should find broad applications in redox biology, chemical biology and the pharmaceutical industry. The protocol for sample preparation takes 3 d, whereas MS measurements and data analyses require 75 min and <30 min, respectively, per sample.
半胱氨酸是所有蛋白质编码氨基酸中独一无二的，这归因于其内在的高亲核性。半胱氨酸巯基基团可以通过广泛的氧化还原机制或各种源自外源性或内源性来源的亲电试剂进行共价修饰。测量蛋白质半胱氨酸对氧化还原扰动或亲电试剂的反应对于理解所涉及的潜在机制至关重要。基于硫醇反应探针的活性蛋白质分析已成为此类分析的首选方法。因此，我们采用了这种方法并开发了一种新的化学生物组学平台，称为“QTRP”（定量硫醇反应分析），该平台依赖于商业上可用的硫醇反应探针 IPM（2-碘-N-（丙-2-炔-1-基）乙酰胺）的能力来共价标记、富集和定量细胞和组织中的反应性半胱氨酸组。在这里，我们提供了 QTRP 的详细和更新的工作流程，其中包括（i）用 IPM 标记细胞或组织样品中的反应性半胱氨酸组（例如，对照与处理）的程序，（ii）将蛋白质样品处理成胰蛋白酶肽，并通过点击化学反应用含有光裂解连接体的同位素标记叠氮生物素试剂标记探针修饰的肽，（iii）用链霉亲和素珠捕获生物素缀合的肽，（iv）通过基于质谱（MS）的鸟枪法蛋白质组学鉴定和定量光释放的肽，以及（v）通过使用蛋白质组学软件 pFind 3 和自动后处理算法简化的信息学管道来解释 MS 数据。我们还在此示例中说明了如何使用 QTRP 挖掘 HO 敏感半胱氨酸并确定复杂蛋白质组中半胱氨酸的固有反应性。我们预计该方案将在氧化还原生物学、化学生物学和制药行业中得到广泛应用。样品制备的方案需要 3 天，而 MS 测量和数据分析分别需要每个样品 75 分钟和<30 分钟。
