Direct identification of intramolecular disulfide links in peptides using negative ion electrospray mass spectra of underivatised peptides. A joint experimental and theoretical study

[MH] − parent anions of underivatised peptides containing an intramolecular disulfide bridge undergo characteristic loss of the elements of H 2 S 2 , a process diagnostic of the presence of the disulfide moeity. This facile process is initiated from a side‐chain enolate anion. Theoretical calculations (at the HF/6‐31G(d)//AM1 level of theory) indicate that the process is exothermic with a small barrier. When the disulfide link involves a C‐terminal Cys, the negative ion spectrum shows an [(MH)(H 2 S 2 +CO 2 )] fragment anion which is usually the main peak of the spectrum. This process is also directed by an enolate anion: theoretical calculations suggest a stepwise sequence with loss of CO 2 preceding loss of H 2 S 2 . Both [(MH)H 2 S 2 ] and [(MH)(H 2 S 2 +CO 2 )] anions undergo backbone cleavage allowing identification of the amino acid sequence of the peptide. Copyright © 2005 John Wiley & Sons, Ltd.