The protonated mercapturic acid conjugate of acrolein, S-(3-oxopropyI)-N-acetyl-L-cysteine (I), undergoes facile retro-Michael loss of acrol ein in the gas phase. To determine whether extensive loss of acrolein would impede structural characterization of acrolein-peptide ad ducts, fragmentation reactions of a series of conjugates, formed by l,4-Michael addition of acrolein to peptides and cysteine derivatives, w ere investigated at collision cell potentials up to -50 V using a triple quadrupole mass spectrometer. Differences in fragmentation dynamics suggest protonation at the sulfur of the N-ac etylcysteine conjugate 1 facilitates retro-Michael elimination of acrolein with a low activation energy relative to other fragmentations. Analogous fragmentation was eliminated after borohydride reduction of the aldehyde to an alcohol. Retro-Michael fragmentation was not significant for acrolein conjugates of glutathione derivatives, suggesting that proton sequestration occurs in peptides with multiple amide linkages even when the peptide does not contain a basic amino group. An unexpected outcome of these experiments was the observation of a facile gas-phase cleavage of peptides on the N-terminal side of S-(3-oxopropylkysteine residues. Such fragmentation behavior may prove useful for locating cysteine residues in peptides. . Protein sulfhydryl groups are especially vulnerable to electrophiIic attack and form thioether conjugates whose presence indicates mechanisms of metabolic activation or detoxification. Knowledge of protein targets and sites of covalent attachment can be used to identify reactive metabolites, and these modified proteins can serve as biomarkers of exposure to xenobiotics in clinical and epidemiological studies [1][2][3][4][5][6][7][8]. One important class of xenobiotic-protein adducts forms via lA-Michael addition of a ,,B-unsaturated carbonyls to nucleophilic amino acid residues. Many a,,B-unsaturated carbonyls including acrylic monomers, combustion by-products or metabolites such as acrolein [1], and endogenous compounds such as 4-hydroxynonenal react with protein nucleophilic side chains via Michael addition [9][10][11].Mass spectrometry has emerged as a powerful analytical tool for the characterization of post-translationally modified peptides and proteins because this approach can identify the modifying group and sites of modification [12]. Earlier studies demonstrated the utility of fast atom bombardment mass spectrometry (FAB/MS) for identification of specific thioether conjuAddress reprint requests to Dr. A. Daniel Jones, Facility for Adv anced Instrumen tation, University of California, Dav is, CA 95616. E-mail: adjones@ucdavis.edu gates from biological samples [3,4,[13][14][15][16][17] . Using FAB or newer ionization techniques coupled with tandem mass spectrometry (MS/MS), researchers determined structures of thioether conjugates extracted from biological matrices [3][4][5][6][7][8][13][14][15][16][17][18][19][20][21][22]. In these MS/MS experiments, structural information was obtained fro...