Electron Capture Dissociation of Gaseous Multiply-Charged Proteins Is Favored at Disulfide Bonds and Other Sites of High Hydrogen Atom Affinity

Abstract

Disulfide bonds in gaseous multiply-protonated proteins are preferentially cleaved in the mass spectrometer by low-energy electrons, in sharp contrast to excitation of the ions by photons or low-energy collisions. For S−S cyclized proteins, capture of one electron can break both an S−S bond and a backbone bond in the same ring, or even both disulfide bonds holding two peptide chains together (e.g., insulin), enhancing the sequence information obtainable by tandem mass spectrometry on proteins in trace amounts. Electron capture at uncharged S−S is unlikely; cleavage appears to be due to the high S−S affinity for H• atoms, consistent with a similar favorability found for tryptophan residues. RRKM calculations indicate that H• capture dissociation of backbone bonds in multiply-charged proteins represents nonergodic behavior, as proposed for the original direct mechanism of electron capture dissociation.

SO2−· electron transfer ion/ion reactions with disulfide linked polypeptide ions

“…The observed loss of only SH 2 might be explained by either a rearrangement to form a monosulfide bond between the two peptide chains, or by the existence of noncovalent bonding between the chains that might hold them together°after°disulfide°bond°cleavage° [43].°The°dearth of backbone cleavages and the relatively abundant chain ions observed in the reaction product ions of Figure°1°indicate°that°the°disulfide°bonds°are°cleaved preferentially to the backbone bonds upon electron transfer in this reaction. Previous work with ECD has also shown a preference for cleavage of the disulfide bond° [24].…”

“…In the ECD work, it was reported that disulfidebound°peptide°chains°cleaved°to°yield°an°odd°electron Chain-S · product, and an even electron Chain-SH product° [24].°The°work°with°ECD°also°reports°that°the°even electron Chain-SH product tends to come from the more highly charged chain, presumably due to polarization of the SOS bond. It has been proposed that this occurs when a hydrogen atom (H · ), generated by electron capture at a protonation site, attacks one of the sulfur atoms, leading to cleavage of the disulfide bond.…”

“…It has been proposed that this occurs when a hydrogen atom (H · ), generated by electron capture at a protonation site, attacks one of the sulfur atoms, leading to cleavage of the disulfide bond. The preference for cleavage of disulfide bonds is then explained by the greater hydrogen affinity of the disulfide°bond° [24],°in°comparison°with°the°peptide°back-bone. Some other studies have indicated that this may not be the only mechanism by which ECD occurs at disulfide linkages, and that direct dissociative electron attachment°may°also°be°a°possibility° [44].°It°is°of°interest to determine if the cleavage observed for electron transfer during reaction with SO 2 Ϫ· yields the same products°as°those°produced°by°ECD.°The°inset°in°Figure 1°shows°an°expanded°view°of°the°region°that°includes the two peptide chains.…”

“…ECD tends to produce more extensive cleavage along the peptide backbone than CID, thereby yielding greater sequence coverage, and often preserves labile post-translational modifications such as phosphorylation and glycosylation [23] when CID does not. An interesting characteristic of ECD is that it has been shown to cleave polypeptide ions preferentially at disulfide bonds [24]. Such distinct fragmentation behaviors of polypeptide ions after capture of an electron make this process particularly interesting and, apparently, complementary to CID.…”

Peptide and protein characterization by high‐rate electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry

“…54 This mechanism can be particularly useful for explanation of the cleavage of disulfide bonds. 54,57 Numerous experimental facts have indicated that the cleavage of the peptide backbone has low specificity and fragmentation of strong bonds can appear in the presence of weak bonds. This non-specific nature of ECD has been explained in terms of the non-ergodicity.…”