SP3‐FAIMS Chemoproteomics for High‐Coverage Profiling of the Human Cysteinome**

Abstract

Chemoproteomics has enabled the rapid and proteome-wide discovery of functional, redox-sensitive, and ligandable cysteine residues. Despite widespread adoption and considerable advances in both sample-preparation workflows and MS instrumentation, chemoproteomics experiments still typically only identify a small fraction of all cysteines encoded by the human genome. Here, we develop an optimized sample-preparation workflow that combines enhanced peptide labeling with singlepot, solid-phase-enhanced sample-preparation (SP3) to improve the recovery of biotinylated peptides, even from small sample sizes. By combining this improved workflow with on-line highfield asymmetric waveform ion mobility spectrometry (FAIMS) separation of labeled peptides, we achieve unprecedented coverage of > 14000 unique cysteines in a single-shot 70 min experiment. Showcasing the wide utility of the SP3-FAIMS chemoproteomic method, we find that it is also compatible with competitive small-molecule screening by isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOP-ABPP). In aggregate, our analysis of 18 samples from seven cell lines identified 34225 unique cysteines using only~28 h of instrument time. The comprehensive spectral library and improved coverage provided by the SP3-FAIMS chemoproteomics method will provide the technical foundation for future studies aimed at deciphering the functions and druggability of the human cysteineome.

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

“…In recent years the scope, scale, and speed with which chemically reactive amino acids can be profiled has accelerated dramatically; chemoselective probes are now available for cysteine 4 , serine 39 , lysine 5 , methionine 6 , tyrosine 7 , aspartate/glutamate 9,40 , tryptophan 41 , and histidine 42 . Supporting this, advances in mass-spectrometry have significantly expanded the number and rate at which individual reactive sites can be profiled 43,44 , and subsequently exploited by electrophilic drug hunters. CORe provides a technology bridge between unbiased proteomic profiling of reactive sites and protein sequence-function relationships, and will be a valuable tool for proteome engineers alongside other methods including MAGE, CRMAGE and CREATE.…”

Enhancing Cysteine Chemoproteomic Coverage Through Systematic Assessment of Click Chemistry Product Fragmentation

“…Samples were prepared as reported. 15 Jurkat proteome (200 μL of 1 mg/mL) was first labeled with IAA 1 or other reagents (4 μL of 100 mM stock solution in DMSO, final concentration = 2 mM) for 1h at ambient temperature. CuAAC was performed with biotin-azide 2 or other reagents (4 μL of 200 mM stock in DMSO, final concentration = 4 mM), TCEP (4 μL of fresh 50 mM stock in water, final concentration = 1 mM), TBTA (12 μL of 1.7 mM stock in DMSO/t-butanol 1:4, final concentration = 100 μM), and CuSO4 (4 μL of 50 mM stock in water, final concentration = 1 mM) for 1h at ambient temperature.…”

“…13,14 However, a central challenge of these studies is that they still only sample a small fraction (2.4%) of all cysteines in the human proteome (estimated to comprise >260,000 residues). 15 Closing this gap requires the development of optimized chemoproteomic sample preparation and data analysis workflows. Supporting this premise, our recent work revealed that using a SP3 cleanup method combined with on-line sample fractionation could achieve 5.5-fold increased coverage compared to prior methods, with decreased instrument time.…”

“…Supporting this premise, our recent work revealed that using a SP3 cleanup method combined with on-line sample fractionation could achieve 5.5-fold increased coverage compared to prior methods, with decreased instrument time. 15 One aspect of global cysteine chemoproteomics workflows that remains largely unexplored is the impact of reagent gas-phase fragmentation during MS/MS analysis and whether such fragmentation might impact data analysis or coverage of labeled peptides. The inertness of the triazole formed during 'click chemistry' to gas-phase reactions has been widely documented.…”

Trendbericht Biochemie: Kovalente Proteinliganden

“…Eine Erhöhung der Zahl quantifizierter Cysteinreste im Proteom haben Keriann Backus und ihre Gruppe erreicht. 3) Neben der kupferkatalysierten Klick-Chemie wurden bisher nur wenige bioorthogonale Reaktionen eingesetzt. Cao und Coautor:innen haben die Suzuki-Miyaura-Reaktion für positionsaufgelöste proteomweite Experimente genutzt.…”