Fragmentation of intra-peptide and inter-peptide disulfide bonds of proteolytic peptides by nanoESI collision-induced dissociation

Abstract

Characterisation and identification of disulfide bridges is an important aspect of structural elucidation of proteins. Covalent cysteine-cysteine contacts within the protein give rise to stabilisation of the native tertiary structure of the molecules. Bottom-up identification and sequencing of proteins by mass spectrometry most frequently involves reductive cleavage and alkylation of disulfide links followed by enzymatic digestion. However, when using this approach, information on cysteine-cysteine contacts within the protein is lost. Mass spectrometric characterisation of peptides containing intra-chain disulfides is a challenging analytical task, because peptide bonds within the disulfide loop are believed to be resistant to fragmentation. In this contribution we show recent results on the fragmentation of intra and inter-peptide disulfide bonds of proteolytic peptides by nano electrospray ionisation collision-induced dissociation (nanoESI CID). Disulfide bridge-containing peptides obtained from proteolytic digests were submitted to low-energy nanoESI CID using a quadrupole time-of-flight (Q-TOF) instrument as a mass analyser. Fragmentation of the gaseous peptide ions gave rise to a set of b and y-type fragment ions which enabled derivation of the sequence of the amino acids located outside the disulfide loop. Surprisingly, careful examination of the fragment-ion spectra of peptide ions comprising an intramolecular disulfide bridge revealed the presence of low-abundance fragment ions formed by the cleavage of peptide bonds within the disulfide loop. These fragmentations are preceded by proton-induced asymmetric cleavage of the disulfide bridge giving rise to a modified cysteine containing a disulfohydryl substituent and a dehydroalanine residue on the C-S cleavage site.

Antimicrobial peptides from the skin of the Asian frog, Odorrana jingdongensis: De novo sequencing and analysis of tandem mass spectrometry data

“…Meanwhile, the cleavages within the intramolecular disulfide loop were rarely found in the positive mode, but were readily observed in the negative mode [41][42][43][53][54][55]. However, the cleavage pathways observed in our study are significantly different from those reported previously.…”

“…Our analysis revealed that there were some unusual fragment ion series, which could be generated by the cleavages at the internal amide bonds or at the C\S bonds on the oxidized Cys within the disulfide loop. Although, these kinds of cleavages rarely occur in the low energy CID MS/MS in positive mode [41][42][43]. The peptide sequence within disulfide loop may be considered as a special cyclic peptide, and its internal amide bond may be cleaved through b x − y z , b x → a x and/or b x → b x − 1 pathways by losing CO (MH + − 28, MH + − X − 28, etc.)…”

“…to produce fragment ion series losing contiguous amino acid residues [38,44]; or its C\S bonds on the oxidized Cys could be cleaved to form a modified Cys residue containing a disulfohydryl substituent (+ SH) and a dehydroalanine residue (− SH). The cleavages caused the formation of the modified b-series ions such as b + 32 (b + SH, β-type) or b − 34 (b − SH, α-type) series ions [43]. The fragment ions, generated by both kinds, or only one kind, or the single pathway of the unusual cleavages were denoted by the italic bold fonts, error 0.0281 Da, 14.9 ppm) and MH + −MG residues (theory mass 1890.0162 Da, error −0.0261 Da, −13.8 ppm), were denoted with superscript 1 and 2, respectively (Fig.…”

“…Moreover, Balaram and Thakur [42] reported fragment ions generated by the unusual cleavages were initiated by the cleavage at Proline within the disulfide loop according to linearized sequence formation mode, however, there is not any Proline residue within the disulfide loop in our studied AMPs. Mormann et al [43]; reported no fragment ion generated by the cleavage at the amide bond within the disulfide loop was observed, in contrast, the cleavages at the internal amide bonds were c l e a v a g e Scheme 1 -Proposed pathways of the unusual cleavages within disulfide loop. The cleavage pathway at internal amide bonds within the disulfide loop are similar to cycle peptide cleavage mode [38,44] followed by secondary cleavage at the C\S bond on oxidized Cys.…”

Gas-phase scrambling of disulfide bonds during matrix-assisted laser desorption/ionization mass spectrometry analysis

“…The same theory was used to explain the fragmentation of intrachain disulfide bonds of peptides by nanoESI CAD and to extend it to a proton-induced asymmetric cleavage of the disulfide bond [22]. Such fragmentations were shown to yield a modified cysteine with a disulfhydryl substituent and a dehydroalanine residue on the C-S cleavage site.…”