Backbone cleavages of [M ¾ H]¾ anions of peptides. New backbone cleavages following cyclisation reactions of a C-terminal [CONH]+G150 group with Ser residues and the use of the γ backbone cleavage initiated by Gln to differentiate between Lys and Gln residues

Abstract

<jats:p> The [M – H]<jats:sup>−</jats:sup> anions of some citropin, caerin and aurein peptides, which contain both Ser residues and a C-terminal CONH<jats:sub>2</jats:sub> functionality, show a number of fragmentations which occur through Ser residues. The first, the loss of CH<jats:sub>2</jats:sub>O from the Ser side chain, always produces pronounced peaks in the spectrum. There are two other fragmentations which are competitive with CH<jats:sub>2</jats:sub>O loss: these involve cleavage of the backbone of the peptide. These fragmentations involve loss of RNH<jats:sub>2</jats:sub> and (RNH<jats:sub>2</jats:sub> + H<jats:sub>2</jats:sub>O) from the [M – H]<jats:sup>−</jats:sup> ion of a peptide of general structure RNHCH(CH<jats:sub>2</jats:sub>OH)CO——NHCHR'CONH<jats:sub>2</jats:sub>. This effectively breaks the peptide backbone between the NH and CH of Ser. We have called these fragmentations γ and ε cleavages, respectively. These processes are initiated by molecular recognition between the C-terminal CONH<jats:sup>−</jats:sup> and the Ser side chain, with the formation of an H-bonded complex between the CONH<jats:sup>−</jats:sup> oxygen and the OH group of Ser. The ion complex, so formed, holds the nucleophilic NH<jats:sup>−</jats:sup> and the electrophilic CH group (of Ser) in positions which allow cyclisation to proceed and produce an intermediate which then initiates those independent processes which involve losses of RNH<jats:sub>2</jats:sub> and (RNH<jats:sub>2</jats:sub> + H<jats:sub>2</jats:sub>O), respectively. These processes have been studied theoretically using a model system at the AM1 level of theory. The isobaric residues Gln and Lys should be distinguishable using negative-ion mass spectrometry, because the Gln residue should effect a γ backbone cleavage, whereas Lys cannot. The study of a number of peptides containing Gln and/or Lys, indicates that this is the case, except when Gln is close to the C-terminal position. The Gln backbone cleavage ions are of small abundance when there are other residues present, for example Asp, which may effect facile backbone cleavage. </jats:p>