Mechanisms for the gas-phase fragmentation reactions of singly and multiply protonated precursor ions of the model S-alkyl cysteine sulfoxide-containing peptides GAILCGAILK, GAILCGAILR, and VTMGHFCNFGK prepared by reaction with iodomethane, iodoacetamide, iodoacetic acid, acrylamide, or 4-vinylpyridine, followed by oxidation with hydrogen peroxide, as well as peptides obtained from an S-carboxyamidomethylated and oxidized tryptic digest of bovine serum albumin, have been examined using multistage tandem mass spectrometry, hydrogen/deuterium exchange and molecular orbital calculations (at the B3LYP/6-31 ϩ G(d,p) level of theory). Consistent with previous reports, CID-MS/MS of the S-alkyl cysteine sulfoxide-containing peptide ions resulted in the dominant "non-sequence" neutral loss of an alkyl sulfenic acid (XSOH) from the modified cysteine side chains under conditions of low proton mobility, irrespective of the alkylating reagent employed. Dissociation of uniformly deuterated precursor ions of these model peptides determined that the loss of alkyl sulfenic acid in each case occurred via a "charge-remote" five-centered cis-1,2 elimination reaction to yield a dehydroalanine-containing product ion. Similarly, the charge state dependence to the mechanisms and product ion structures for the losses of CO 2 , CO 2 ϩ H 2 O and CO 2 ϩ CH 2 O from S-carboxymethyl cysteine sulfoxide-containing peptides, and for the losses of CH 2 CHCONH 2 and CH 2 CHC 5 H 4 N, respectively, from S-amidoethyl and S-pyridylethyl cysteine sulfoxide-containing peptide ions have also been determined. The results from these studies indicate that both the proton mobility of the peptide precursor ion and the nature of the S-alkyl substituent have a significant influence on the abundances and charge states of the product ions resulting from the various competing fragmentation pathways. (J Am Soc Mass Spectrom 2007, 18, 1690 -1705) © 2007 American Society for Mass Spectrometry C onventional approaches for mass spectrometry based protein identification and characterization typically involve the reduction and alkylation of cysteine residues before enzymatic digestion [1][2][3][4]. Numerous reports in the literature have demonstrated that the resulting thio-ether bonds are susceptible to oxidation, either during the enzymatic digestion process or subsequent sample handling steps, before mass spectrometry analysis [5][6][7][8]. These reports have also demonstrated that fragmentation of the resultant S-alkyl cysteine sulfoxide-containing peptides under lowenergy collision induced dissociation (CID)-tandem mass spectrometry (MS/MS) conditions can result in the formation of abundant product ions via cleavages occurring within the modified cysteine side chains [5][6][7][8]. While these "non-sequence" ions are indicative of the presence of the modified amino acid residue within the peptide, their formation at high abundance may "suppress" the formation of desired "sequence" ion information, thereby limiting the utility of "de novo" analysis strategies [9] or...