Exploring the Potential of Ultrafast Arylation for Capping Cysteine Residues with Fixed Charge Modifications

Reduction of disulfide bonds in proteins followed by selective modification of Cysteine (Cys) residues is a common practice in proteomics experiments to facilitate digestion into peptides and ultimately identification by mass spectrometry (MS). Due to its high nucleophilicity, a variety of reaction pathways can be used to block Cys; however, arylation is not often employed. Recently, Lipka and co-workers reported an electrophilic Cys arylation reagent, <i>N</i>-methyl-2-methylsulfonylpyridinium (CAP4), that can arylate free thiols with high selectivity and rate (Lipka, B. M.; Honeycutt, D. S.; Bassett, G. M.; Kowal, T. N.; Adamczyk, M.; Cartnick, Z. C.; Betti, V. M.; Goldberg, J. M.; Wang, F. Ultra-Rapid Electrophilic Cysteine Arylation. J. Am. Chem. Soc. 2023, 145 (43), 23,427-23,432). Additionally, CAP4 modification includes the addition of a fixed charge to the side chain that could potentially influence MS experiments. The fixed charge could be useful if it affords better sensitivity or detrimental if it negatively affects fragmentation. Herein, we show that CAP4 modification leads to favored fragmentation pathways involving the modified side chain in both higher-energy collisional dissociation (HCD) and electron-transfer dissociation (ETD). Abundant side chain losses from CAP4 are observed in HCD, while in ETD, electron transfer leads to the formation of a hydrogen deficient beta radical at the Cys residue. Formation of the beta radical is shown to be charge-state dependent, and collisional activation of the radical leads to radical-directed dissociation (RDD)-derived fragments. In addition, we evaluated the potential of CAP4 in a bottom-up proteomics workflow using a model protein mixture. Despite its influence on fragmentation, rapid arylation (5 min) by CAP4 resulted in comparable sequence coverage and total modified Cys-containing peptide IDs when compared to 1 h iodoacetamide modification.