Characterization of benzoquinone-peptide adducts by electrospray mass spectrometry

Benzoquinone adducts were prepared with model peptides to identify characteristic features of adduct fragmentation in tandem mass spectrometry (MS) experiments. Model peptides contained cysteine and had a molecular mass of less than 2 kDa to facilitate peptide fragmentation in tandem MS analyses. Peptides were adducted with an excess of benzoquinone, and the adducts were analyzed by LC/MS. Adducts were identified by addition of 108 Da to the monoisotopic mass of the peptide, except in the case of oxytocin, which formed a bis adduct with addition of 216 Da. Tandem MS experiments were performed on the [M + 2H](2+) ions and/or the [M + H](+) ions. Sequence information obtained from modified peptides was comparable to that of their unmodified counterparts. A unique ion pair separated by 141 or 142 Da corresponding to beta-elimination of benzoquinol-S or benzoquinol-SH from a b(n) or y(n) series ion indicated attachment at the sulfur of the cysteine residue. An alternate ion pair of 211 Da corresponded to fragmentation at the peptide bond on either side of the adducted cysteine. Enzymatic digestion of BSA and a 2560 Da frog peptide with trypsin yielded tryptic peptides, which were treated with benzoquinone. In addition to ion pairs of 142 and 211 Da, singly and doubly charged tryptic peptide adducts showed a neutral loss of 142 Da from the precursor. Either one or both ion pairs were present in more than half of all the peptides that were examined. The neutral loss of 142 Da was present in all singly charged tryptic peptide adducts and in 11 out of 14 doubly charged tryptic peptide adducts. The data indicate that reliable detection of benzoquinone-cysteinyl peptide adducts requires monitoring of multiple spectral characteristics.