No abstract

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 (PDMs) that can be automatically recognized by pChem, with masses accurately calculated and sites precisely localized. Further, 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. Of note, the analyses reveal that the formation of unexpected PDMs can be driven by endogenous reactive metabolites (e.g., bioactive aldehydes and glutathione). Together, pChem is a powerful and user-friendly tool that aims to facilitate the development of probes for the ever-growing field of chemoproteomics.Chemical probe coupled with mass spectrometry (MS)-based proteomics, herein termed chemoproteomics, offers versatile tools to globally profile protein features and to systematically interrogate the mode of action of small molecules in a native biological system 1 . For instance, bioorthogonal probes surrogating endogenous metabolites (e.g., sugars and lipids) enable the proteome-wide mapping of post-translational modifications (PTMs) on specific amino acid residues 2 . In addition, various activity-based protein profiling (ABPP) probes have been developed by targeting amino acid residues including cysteine 3, 4 , lysine 5 , tyrosine 6 , methionine 7, 8 , histidine 9 , aspartate and glutamate 10,11 , as well as their PTM forms [12][13][14] , which greatly expand the chemical space in complex proteomes for probing fundamental biology and for discovering new small molecule-based therapies.Nonetheless, the development of an efficient and selective probe for chemoproteomics can still be challenging. It is particularly difficult to unbiasedly assess its chemoselectivity at a proteome-wide scale, since a chemical probe displaying selectivity well-characterized in vitro would possibly generate unexpected modifications owing to potential cross-reactivity in complex biological systems. In addition, unforeseeable probe-derived modifications (PDMs) may be yielded during sample preparation or in-source MS fragmentation, thereby causing inhomogeneous modifications on the same sites and

We report a modification-centric, blind-search tool termed pChem to provide a streamlined pipeline for unbiased assessing of the performance of chemoproteomic probes. The pipeline starts with an experimental setting for isotopically coding probe-derived modifications that can be automatically recognized, accurately calculated and precisely localized by pChem with neither prior knowledge nor manual inspection. Further, 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 various bioorthogonal probes, including 15 activity-based protein profiling (ABPP) probes and 3 metabolic labeling probes. Together, pChem is a user-friendly computational tool for probe developers, even those with no experience in informatics, and aims to facilitate the development and optimization of probes for the ever-growing field of chemoproteomics.