SP3-FAIMS Chemoproteomics for High Coverage Profiling of the Human Cysteinome

Abstract

<p>We report a new cysteine chemoproteomic method, termed SP3-FAIMS chemoproteomics, which enables rapid and high coverage analysis of the human cysteinome. By combining enhanced cysteine biotinylation with SP3 sample decontamination and FAIMS online fraction, we identified in aggregate 34,225 unique cysteines found on 7,243 proteins. Showcasing the versatility of our method, integration with the isoTOP-ABPP workflow enabled the high throughput discovery of cysteines labelled by electrophilic compounds. </p>

Reimagining Druggability Using Chemoproteomic Platforms

“…Evaluating probe-modified peptides identified in chemoproteomic experiments of many laboratories in the chemical biology field reveals tens of thousands of nucleophilic sites that have been modified by the array of existing reactivity-based probes, with the potential to capture hundreds of thousands of sites in the future. − ,− While not all of these sites represent binding pockets, there are likely thousands of sites within these data that represent potential ligandable hotspots that can be interrogated by a suitable electrophile, making chemoproteomic approaches a powerful entry point for discovery of ligandable sites and new covalent small-molecule binders against targets of interest. Discovery of new ligandable sites is especially significant in light of the new wave of bispecific therapies, which can convert noninhibitory ligands into consequential binders by recruiting effector proteins.…”

“…The ABPP method also continues to evolve with advances in sample preparation, separation of peptides, mass spectrometry capabilities, and bioinformatics. A recent study by Yan et al combines optimized biotin conjugation methods and solid-phase SP3 extraction methods with field asymmetric ion mobility mass spectrometry (FAIMS) and the Thermo trihybrid Eclipse mass spectrometer to identify >15 000 unique cysteines, greatly increasing coverage of the cysteine proteome …”

Chemoproteomic Profiling by Cysteine Fluoroalkylation Reveals the DNA Repair Protein XRCC5 as a Functional Target of Myrocin G

“…This time we identified 7818 adducts with 1 whereas 7 modified 10062 cysteines, showing a ~29% improvement compared to 1 (Figure 3D and Tables S7 and S8). We speculate that this number could be further improved by solid-phase enhanced sample preparation 51 and sample fractionation. 15 It is worth mentioning that 7 retained excellent cysteine selectivity (96.5%) even at this high concentration (Figure 3C) and that the average area of 7-modified peptides remained ~2.5-fold higher than the area of 1-modified peptides (Figure 3B).…”

Comprehensive Overview of Bottom-Up Proteomics Using Mass Spectrometry

“…Protein clean-up approaches for glycoproteomics may differ from other proteomics experiments because glycopeptides are more hydrophilic than most peptides. Some approaches mentioned in the literature include: filter-aided sample preparation (FASP), suspension traps (S-traps), and protein aggregation capture (PAC). ,− Multiple proteases may be used to increase the sequence coverage and detect more modification sites, such as: trypsin, chymotrypsin, pepsin, WaLP/MaLP, GluC, AspN, pronase, proteinase K, OgpA, StcEz, BT4244, AM0627, AM1514, AM0608, Pic, ZmpC, CpaA, IMPa, PNGase F, Endo F, Endo H, and OglyZOR . Mass spectrometry has improved over the past decade, and now many strategies are available for glycoprotein structure elucidation and glycosylation site quantification .…”