A modification-centric assessment tool for the performance of chemoproteomic probes

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

Comparative analysis of chemical cross-linking mass spectrometry data indicates that protein STY residues rarely react with N-hydroxysuccinimide ester cross-linkers

“…Therefore, most if not all STY-cross-link identification are unreliable. This is consistent with a recent finding that a pair of light/heavy isotope-labeled NHS ester probes did not label protein/ peptides at STY residue 46 . It is also supported by an earlier finding that at pH 6.7 and pH 7.8, no detectable reaction products were found between an NHS ester cross-linker and peptide hydroxyl groups of STY residues, even after 24 hours 30 .…”

“…In agreement with the above conclusion, a recent proteome-scale study has found no evidence of a NHS ester probe targeting S, T, or Y 46 . In this study, a HeLa cell lysate was incubated with a pair of light and heavy isotope encoded NHS ester probe, and pChem-a software tool developed specifically to assign modification sites of chemical probes-was employed to analyze the data.…”

Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

“…Here we envisioned combining proximity labeling via the ultra-fast biotin ligase TurboID with cysteine chemoproteomics 11,21,[24][25][26][27][28][29][30]57,58 to enable fractionation-free capture of the subcellular cysteinome, for both residue identification and quantification of cysteine oxidation. We were inspired by recent reports of two-step capture for subcellular phosphoproteomics, in which proteins biotinylated by TurboID were first enriched on avidin resin followed by peptide-level capture of phosphopeptides 59 .…”

“…Application of these methods have pinpointed cysteines differentially oxidized in association with high levels of ROS and RNS, such as those of TRX 13,14 , GAPDH 15,16 and HBB 17 . Given the recent advent of cysteine-reactive small molecules as precision therapies for the treatments of cancers and immune disorders [18][19][20] , cysteine chemoproteomic methods have also emerged as enabling technology for pinpointing ligandable or potentially 'druggable' residues proteome-wide [21][22][23][24][25][26][27][28][29][30] . A central remaining challenge for these studies is the lack of a priori knowledge about the functional impact of covalent modification.…”

Protein–RNA interactions: from mass spectrometry to drug discovery

“… The RBR-ID method does not include a dedicated enrichment step, but compares peptide intensities between cross-linked samples prepared with 4-thiouridine(4-SU)-labelled RNA and non-cross-linked samples. The mass shift introduced by covalently bound RNA will lead to a decrease in signal for successfully cross-linked peptides between the two samples [ 15 ]. 2C (silica-based solid-phase extraction), TRAPP (total RNA-associated protein purification), and RBS-ID utilise silica beads/membranes for nucleic acid enrichment [ 16 , 17 ].…”