Activity-based protein profiling (ABPP) has emerged as a powerful functional chemoproteomic strategy which enables global profiling of proteome reactivity toward bioactive small molecules in complex biological and/or pathological processes. To quantify the degree of reactivity in a site-specific manner, an isotopic tandem orthogonal proteolysis (isoTOP)-ABPP strategy has been developed; however, the high cost and long workflow associated with the synthesis of isotopically labeled cleavable tags limit its wide use. Herein, we combined reductive dimethyl labeling with TOP-ABPP to develop a fast, affordable, and efficient method, termed “rdTOP-ABPP”, for quantitative chemical proteomics with site-specific precision and triplex quantification. The rdTOP-ABPP method shows high accuracy and precision, good reproducibility, and better capacity for site identification and quantification and is highly compatible with many commercially available cleavable tags. We demonstrated the power of rdTOP-ABPP by profiling the target of (1S,3R)-RSL3, a canonical inducer for cell ferroptosis, and provided the first global portrait of its proteome reactivity in a quantitative and site-specific manner.

中文翻译：

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基于二甲基标签的针对特定地点定量化学蛋白质组学的策略

基于活动的蛋白质谱分析（ABPP）已成为一种功能强大的化学趋化策略，可以对复杂的生物和/或病理过程中的蛋白质组反应性对生物活性小分子进行全局谱分析。为了以位点特异性的方式量化反应程度，已经开发了同位素串联正交蛋白水解（isoTOP）-ABPP策略。然而，与同位素标记的可裂解标签的合成相关的高成本和长工作流程限制了其广泛的用途。在本文中，我们将还原性二甲基标记与TOP-ABPP结合在一起，开发了一种快速，负担得起且高效的方法，称为“ rdTOP-ABPP”，用于具有特定位点精度和三重定量的定量化学蛋白质组学。rdTOP-ABPP方法显示出高精度和高精度，良好的重现性，具有更好的位点识别和定量能力，并且与许多市售可裂解标签高度兼容。我们通过剖析（1S，3 R）-RSL3，细胞肥大症的典型诱导剂，以定量和位点特异性的方式提供了其蛋白质组反应性的第一张全貌。