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 H₂O₂-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.

由于其固有的高亲核性，半胱氨酸在所有蛋白质编码氨基酸中是独一无二的。半胱氨酰硫醇基团可以通过广泛的氧化还原机制或通过源自外源或内源的各种亲电子试剂进行共价修饰。测量蛋白质半胱氨酸对氧化还原扰动或亲电子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基于活性的蛋白质分析一直是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蛋白质组学平台，称为“QTRP”（定量硫醇反应性分析），它依赖于市售硫醇反应探针 IPM（2-碘-N）的能力-(prop-2-yn-1-yl)acetamide) 共价标记、富集和量化细胞和组织中的反应性半胱氨酸组。在这里，我们提供了详细和更新的 QTRP 工作流程，其中包括 (i) 使用 IPM 标记细胞或组织样本中的反应性半胱氨酸组（例如，对照与治疗），(ii) 将蛋白质样本加工成胰蛋白酶肽并标记通过点击化学反应，用含有光可裂解接头的同位素标记叠氮基生物素试剂的探针修饰肽，（iii）用链霉亲和素珠捕获生物素偶联肽，（iv）通过质谱法鉴定和量化光释放的肽（基于 MS) 的鸟枪蛋白质组学和 (v) 使用蛋白质组学软件 pFind 3 和自动后处理算法通过简化的信息管道解释 MS 数据。₂ O ₂敏感的半胱氨酸，用于确定复杂蛋白质组中半胱氨酸的内在反应性。我们预计该协议应该在氧化还原生物学、化学生物学和制药工业中得到广泛应用。样品制备协议需要 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。