Profiling the Proteome-Wide Selectivity of Diverse Electrophiles

Abstract

<p><a>Targeted covalent inhibitors are powerful entities in drug discovery, but their application has so far mainly been limited to addressing cysteine residues. The development of cysteine-directed covalent inhibitors has largely profited from determining their proteome-wide selectivity using competitive residue-specific proteomics. Several probes have recently been described to monitor other amino acids using this technology and many more electrophiles exist to modify proteins. Nevertheless, a direct, proteome‑wide comparison of the selectivity of diverse probes is still entirely missing. Here, we developed a completely unbiased workflow to analyse electrophile selectivity proteome‑wide and applied it to directly compare 54 alkyne probes containing diverse reactive groups. In this way, we verified and newly identified probes to monitor a total of nine different amino acids as well as the <i>N</i>‑terminus proteome‑wide. This selection includes the first probes to globally monitor tryptophans, histidines and arginines as well as novel tailored probes for methionines, aspartates and glutamates.</a></p>

SP3-Enabled Rapid and High Coverage Chemoproteomic Identification of Cell-State–Dependent Redox-Sensitive Cysteines

“…Critical to the success of the SP3-Rox method was our application of the FragPipe computational pipeline for highly sensitive peptide identification, as well as rigorous vetting of the FragPipe's built-in quantification module IonQuant by comparison with Skyline quantification. While proteomics tools such as FragPipe and Skyline have been widely adopted by the proteomics community, their use in chemoproteomics and redox proteomics has been limited to a handful of examples ( 40 , 88 , 89 ). Building upon our prior findings that MSFragger is compatible with search of chemoproteomics data generated using a FAIMS device for online fractionation, we first confirmed that search of samples labeled with the heavy and light IAA probes at a 1:1 stoichiometry afforded comparable PSMs for light- and heavy-labeled peptides.…”

Advances in covalent drug discovery

“…However, sulfonyl fluoride-based probes are not completely selective for lysine and have also been used to target tyrosine residues 209 . A recent preprint reported the use of a chemoproteomic approach to profile the amino acid reactivity preference of 54 different electrophiles, which will prove to be a great resource for covalent ligand discovery 210 . Combining comprehensive electrophile profiling, lysine-directed chemoproteomics and structure-guided approaches will enable scientists to leverage the abundance of lysine residues adjacent to ligand binding sites to enhance covalent drug discovery.…”

Eukaryotic catecholamine hormones influence the chemotactic control of Vibrio campbellii by binding to the coupling protein CheW

“…3A) (40). In an unbiased analysis (41), we detected modified peptides with the added mass of the adduct with PE-P plus light or heavy isoDTB tag, respectively (Fig. 3B).…”

“…Analysis of the modifications introduced by PE-P proteome-wide. (A) Workflow applied to study binding mode and sites of PE-P using isotopically labeled desthiobiotin (isoDTB) tags(39,41). V. campbellii lysate was treated with 10 µM PE-P (blue circle), irradiated, split, and subjected to CuAAC with either light-(turquoise rectangle) or heavy-labeled (purple rectangle) isoDTB azide.…”