Mechanistic Insights into an Unusual Side-Chain-Mediated N–C^α Bond Cleavage under Collision-Induced Dissociation Conditions in the Disulfide-Containing Peptide Conopressin

Abstract

Conopressin, a nonapeptide disulfide CFIRNCPKG amide present in cone snail venom, undergoes a facile cleavage at the Cys6−Pro7 peptide bond to yield a disulfide bridged b 6 ion. Analysis of the mass spectral fragmentation pattern reveals the presence of a major fragment ion, which is unambiguously assigned as the tripeptide sequence IRN amide. The sequence dependence of this unusual fragmentation process has been investigated by comparing it with the fragmentation patterns of related peptides, oxytocin (CYIQNCPLG amide), Lys-vasopressin (CYFQNCPKG amide), and a series of synthetic analogues. The results establish the role of the Arg4 residue in facilitating the unusual N−C α bond cleavage at Cys6. Structures are proposed for a modified disulfide bridged fragment containing the Cys1 and Cys6 residues. Gas-phase molecular dynamics simulations provide evidence for the occurrence of conformational states that permit close approach of the Arg4 side chain to the Cys6 C β methylene protons.

Mass spectral and theoretical investigations of N–Cαbond cleavages in the disulfide‐containing peptide TTCPYCKK and its analogues

“…Recently, Kumar et al investigated fragmentations of disulfide‐containing nonapeptide conopressin (CFIRNCPKG‐NH 2 ) and its peptide analogues (CFIRDCPKG‐NH 2 , CFIRACPLG‐NH 2 , CYIQNCPLG‐NH 2 and CYFQNCPKG‐NH 2 ) 23 . When the sequences contained an Arg residue, an unusual N–C α bond cleavage was observed and the fragment [C 1 Y 2 |C 6 M ] was afforded (where “|” denotes the linking bond with former part and C M denotes an modified Cys; cf.…”

“…Scheme 1, structure I ), indicating the critical role of R 4 in the facilitation of the N–C α bond cleavage at C 6 residue. Canonical peptide bond and the unusual N–C α bond cleavages occurred together only in the Arg‐containing nonapeptide (CFIRNCPKG‐NH 2 ) and its peptide analogues (CFIRDCPKG‐NH 2 and CFIRACPLG‐NH 2 ) under CID conditions 23 . Usually, the specially modified disulfide fragment of [C 1 F 2 |C 6 M ] was identified as a persulfide, which contained the Cys residue (C 1 ) and a truncated unit of C 6 residue.…”

“…Isomeric structures: I and II were adopted from Kumar et al, 23 while III and IV are proposed ions from the present study. [Color figure can be viewed at wileyonlinelibrary.com]…”

“…Isomeric structures: I and II were adopted from Kumar et al,23 while III and IV are proposed ions from the present study. [Color figure can be viewed at wileyonlinelibrary.com] T A B L E 1 Amino acid sequences of thiol-disulfide that contain proline Sequence T 1 T 2 C 3 P 4 X 5 C 6 K 7 K 8 X = Y, S, T and p Y Ac-T 1 T 2 C 3 P 4 X 5 C 6 K 7 K 8 -NH 2 X = Y, S, T and p Y T 1 T 2 C 3 P 4 p X 5 C 6 K 7 K 8 X = Y, S, T and p Y Ac-T 1 T 2 C 3 P 4 p X 5 C 6 K 7 K 8 -NH 2 X = Y, S, T and p Y Note: p X 5 denotes the phosphorylated side chains of X 5 .…”

“…Usually, the specially modified disulfide fragment of [C 1 F 2 jC 6 M ] was identified as a persulfide, which contained the Cys residue (C 1 ) and a truncated unit of C 6 residue. However, this disulfide-linked C 1 -C 6 was also found to appear in the nonapeptide of CFIVACPLG-NH 2 without Arg residue.The absence or presence of the unusual N-C α bond cleavage at C 6 residue was supposed to rely on the appearance of C-terminus amidated tripeptide of IRX-NH 2 (X = N, D and A) in the mass spectra 23. However, if the number of the residues between the two Cys was reduced, C-terminus amidated tripeptide analogues might not be considered as a criterion to justify the ways of bond cleavage.Besides, the analogous cystine fragment linked to Y 2 [C 1 MA Y 2 jC 6 MB ] (C 1 MA = À17, C 6 MB = À28) at m/z 323 was suggested in both cases (cf.…”