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

Chang Ho Sohn, Jinshan Gao, Daniel A. Thomas, Tae Young Kim, William A. Goddard, J. L. Beauchamp

Research output: Contribution to journalArticleResearchpeer-review

12 Citations (Scopus)

Abstract

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 (SH2). 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.

Original languageEnglish
Pages (from-to)4550-4560
Number of pages11
JournalChemical Science
Volume6
Issue number8
DOIs
StatePublished - 1 Aug 2015

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Disulfides
Free Radicals
Mass spectrometry
Insulin
Peptides
Carbon
Hydrogen
Activation energy
Chemical activation
Ions
Sulfur
Proteins

Cite this

Sohn, Chang Ho ; Gao, Jinshan ; Thomas, Daniel A. ; Kim, Tae Young ; Goddard, William A. ; Beauchamp, J. L. / Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry. In: Chemical Science. 2015 ; Vol. 6, No. 8. pp. 4550-4560.
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abstract = "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 (SH2). 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.",
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Mechanisms and energetics of free radical initiated disulfide bond cleavage in model peptides and insulin by mass spectrometry. / Sohn, Chang Ho; Gao, Jinshan; Thomas, Daniel A.; Kim, Tae Young; Goddard, William A.; Beauchamp, J. L.

In: Chemical Science, Vol. 6, No. 8, 01.08.2015, p. 4550-4560.

Research output: Contribution to journalArticleResearchpeer-review

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AU - Sohn, Chang Ho

AU - Gao, Jinshan

AU - Thomas, Daniel A.

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AU - Beauchamp, J. L.

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N2 - 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 (SH2). 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.

AB - 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 (SH2). 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.

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