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Published August 2015 | Published + Supplemental Material
Journal Article Open

Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry


We investigate the mechanism of disulfide bond cleavage in gaseous peptide and protein ions initiated by a covalently-attached regiospecific acetyl radical using mass spectrometry (MS). Highly selective S–S bond cleavages with some minor C–S bond cleavages are observed by a single step of collisional activation. We show that even multiple disulfide bonds in intact bovine insulin are fragmented in the MS2 stage, releasing the A- and B-chains with a high yield, which has been challenging to achieve by other ion activation methods. Yet, regardless of the previous reaction mechanism studies, it has remained unclear why (1) disulfide bond cleavage is preferred to peptide backbone fragmentation, and why (2) the S–S bond that requires the higher activation energy conjectured in previously suggested mechanisms is more prone to be cleaved than the C–S bond by hydrogen-deficient radicals. To probe the mechanism of these processes, model peptides possessing deuterated β-carbon(s) at the disulfide bond are employed. It is suggested that the favored pathway of S–S bond cleavage is triggered by direct acetyl radical attack at sulfur with concomitant cleavage of the S–S bond (S_H2). The activation energy for this process is substantially lower by ~9–10 kcal mol^(−1) than those of peptide backbone cleavage processes determined by density functional quantum chemical calculations. Minor reaction pathways are initiated by hydrogen abstraction from the α-carbon or the β-carbon of a disulfide, followed by β-cleavages yielding C–S or S–S bond scissions. The current mechanistic findings should be generally applicable to other radical-driven disulfide bond cleavages with different radical species such as the benzyl and methyl pyridyl radicals.

Additional Information

© 2015 The Royal Society of Chemistry. This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Received 10 Apr 2015, Accepted 20 May 2015. First published online 20 May 2015. This work was supported by the National Science Foundation through grant CHE-0416381 and the Resource Center for Mass Spectrometry in the Beckman Institute at the California Institute of Technology. C.H.S. acknowledges a fellowship from the Kwanjeong Educational Foundation. Electron capture dissociation was performed using the LTQ-FT in the Beckman Institute Proteome Exploration Laboratory. Computational resources were kindly provided by the Materials and Process Simulation Center at the California Institute of Technology.

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August 20, 2023
August 20, 2023