Chemoproteomics has emerged as a key technology to expand the functional space in complex proteomes for probing fundamental biology and for discovering new small-molecule-based therapies. Here we report a modification-centric computational tool termed pChem to provide a streamlined pipeline for unbiased performance assessment of chemoproteomic probes. The pipeline starts with an experimental setting for isotopically coding probe-derived modifications that can be automatically recognized by pChem, with masses accurately calculated and sites precisely localized. pChem exports on-demand reports by scoring the profiling efficiency, modification homogeneity and proteome-wide residue selectivity of a tested probe. The performance and robustness of pChem were benchmarked by applying it to eighteen bioorthogonal probes. These analyses reveal that the formation of unexpected probe-derived modifications can be driven by endogenous reactive metabolites (for example, bioactive aldehydes and glutathione). pChem is a powerful and user-friendly tool that aims to facilitate the development of probes for the ever-growing field of chemoproteomics.

化学蛋白质组学已成为扩展复杂蛋白质组功能空间以探索基础生物学和发现新的基于小分子的疗法的关键技术。在这里，我们报告了一种名为 pChem 的以修改为中心的计算工具，为化学蛋白质组学探针的无偏性能评估提供了一个简化的流程。该管道从同位素编码探针衍生修饰的实验设置开始，pChem 可以自动识别该修饰，精确计算质量并精确定位位点。pChem 通过对测试探针的分析效率、修饰同质性和蛋白质组范围内的残基选择性进行评分来按需输出报告。pChem 的性能和稳健性通过将其应用于 18 个生物正交探针来进行基准测试。这些分析表明，意外的探针衍生修饰的形成可以由内源性反应性代谢物（例如，生物活性醛和谷胱甘肽）驱动。pChem 是一种功能强大且用户友好的工具，旨在促进为不断发展的化学蛋白质组学领域开发探针。