Characterization of benzoquinone-peptide adducts by electrospray mass spectrometry

adduct fragmentation in tandem mass spectrometry (MS) experiments. Model pe ptides contained cysteine and had a molecular mass of less than 2 kDa to fa cilitate peptide fragmentation in tandem MS analyses. Peptides were adducte d with an excess of benzoquinone, and the adducts were analyzed by LC/MS. A dducts were identified by addition of 108 Da to the monoisotopic mass of th e peptide, except in the case of oxytocin, which formed a bis adduct with a ddition of 216 Da. Tandem MS experiments were performed on the [M + 2H](3+) ions and/or the [M + H](+) ions. Sequence information obtained from modifi ed peptides was comparable to that of their unmodified counterparts. A uniq ue ion pair separated by 141 or 142 Da corresponding to P-elimination of be nzoquinol-S or benzoquinol-SH from a b(n) or y(n) series ion indicated atta chment 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 wi th trypsin yielded tryptic peptides, which were treated with benzoquinone. In addition to ion pairs of 142 and 211 Da, singly and doubly charged trypt ic peptide adducts showed a neutral loss of 142 Da from the precursor. Eith er 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 c harged tryptic peptide adducts and in 11 out of 14 doubly charged tryptic p eptide adducts. The data indicate that reliable detection of benzoquinone-c ysteinyl peptide adducts requires monitoring of multiple spectral character istics.