Fragmentation of Protonated Thioether Conjugates of Acrolein Using Low Collision Energies Christa H. Oberth and A. Daniel Jones Facility for Advanced Instrumentation, University of California, Davis, California, USA The protonated mercapturic acid conjugate of acrolein, S-(3-oxopropyI)-N-acetyl-L-cysteine (I), undergoes facile retro-Michael loss of acrolein 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, were 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-acetylcysteine 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. (J Am Soc Mass Spectrom 1997, 8, 727-736) © 1997 American Society for Mass Spectrometry ovalent modification of proteins and DNA by gates from biological samples [3,4, 13-17]. Using FAB reactive electrophiles such as xenobiotic metab­ or newer ionization techniques coupled with tandem Colites can lead to cancer or cell death [1]. Protein mass spectrometry (MS/MS), researchers determined sulfhydryl groups are especially vulnerable to electro­ structures of thioether conjugates extracted from bio­ phiIic attack and form thioether conjugates whose pres­ logical matrices [3-8, 13-22]. In these MS/MS experi­ ence indicates mechanisms of metabolic activation or ments, structural information was obtained from colli­ detoxification. Knowledge of protein targets and sites of sion induced dissociation (CIO). In fact, Murphy et al. covalent attachment can be used to identify reactive [20] concluded that fragmentation patterns of thioether metabolites, and these modified proteins can serve as conjugates have much in common, but that universal biomarkers of exposure to xenobiotics in clinical and recognition of class characteristic ions has been uncer­ epidemiological studies [1-8]. One important class of tain . xenobiotic-protein adducts forms via lA-Michael addi­ More detailed information regarding activation en­ tion of a,,B-unsaturated carbonyls to nucleophilic amino ergies for specific fragmentation reactions can be ob­ acid residues. Many a,,B-unsaturated carbonyls includ­ tained by generating energy resolved product ion spec­ ing acrylic monomers, combustion by-products or me­ tra. Energy resolved product ion spectra are acquired tabolites such as acrolein [1], and endogenous com­ by making incremental changes in collision cell poten­ pounds such as 4-hydroxynonenal react with protein tial and determining the dependence of product ion nucleophilic side chains via Michael addition [9-11]. abundances on the translational energy of precursor Mass spectrometry has emerged as a powerful ana­ ions [23, 24]. At the lowest collision energies, only lytical tool for the characterization of post-translation­ fragmentation reactions with the lowest activation en­ ally modified peptides and proteins because this ap­ ergies are accessible. Incremental increases in collision proach can identify the modifying group and sites of energy provide access to reaction pathways leading to modification [12]. Earlier studies demonstrated the util­ more and different product ions [23-28]. Normalized ity of fast atom bombardment mass spectrometry product ion abundances can be plotted to show their (FAB/MS) for identification of specific thioether conju- dependence upon collision cell potential. These graphs are termed energy resolved dissociation curves, and they assist efforts to optimize CIO experiments aimed at Address reprint requests to Dr. A. Daniel Jones, Facility for Adv anced structure determination or specific detection. Subtle Instrumen tation, University of California, Davis, CA 95616. E-mail: [email protected] differences in fragmentation dynamics are easily stud- © 1997 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received July 15, 1996 1044-0305/ 97/51 7.00 Revised March 12, 1997 PI! 51044-0305(97)00032-9 Accepted March 13, 1997 728 OBERTH AND JONES J Am Soc Mass Spectrom 1997, 8, 727-736 Experimental I. R, = -CH,CH,CHO } Materials II. R, = .CH,CII,CH,OH { III. R, = .CH,CII,CII=NOH Caution: Acrolein is a potent and toxic lachrymator and should be handled ill a fume hood to minimize exposure. Sodium borohydride is a toxicflammable solid and should be Y2 Z2 Y, handled carefully. IV. R, = -CH,CH,CHO } N-Acety1cysteine, glutathione, and oxidized gluta­ -c~t~r~-,~ V. R, = ·CH,CII,CH,OIl thione (GSSG) were obtained from Sigma (S1. Louis, .. { VI. R, = -CH,CH,CH=NOH SR,~ MO). Acrolein, sodium borohydride (NaBH4), hydrox­ ~ a, RI( ylamine hydrochloride, trifluoroacetic acid (TFA), and Sephadex QAE-A-50, were obtained from Aldrich (Mil­ V2 Z2 Y, waukee, WI). Acetic anhydride, acetonitrile, and dithio­ JVII. R. = ·H } threitol were from Fisher (Pittsburgh, PA). Bond-Elut )-~d~jdL-=" \ VIII. R, = .CH,CH,CHO CBA solid phase extraction cartridges were from Ana­ SRx~ Iytichem (Varian, Harbor City, CA). All water used in ,------T- the following procedures was obtained from a Barn­ Rx a, steadlThermolyne (Dubuque, IA) Nanopure water fil­ tration system. Synthesis g h Syntheses of conjugates were conducted by adding Figure 1. Structures of thioether conjugates used in this study. acrolein to solutions of various thiols, flushing the headspace of the reaction vials to remove oxygen, and ied by using tandem quadrupole instruments where keeping reaction vials wrapped in aluminum foil to ion-molecule collisions are low energy processes [23-25, prevent photochemical degradation. Reaction progress 27-331. In such experiments, most fragmentation is was monitored by using free zone capillary electro­ charge directed as fragmentation barriers are lowered phoresis (CE) with spectrophotometric detection at 214 by a nearby positive charge. nm. When reactions were complete, products were Electrospray and FAB ionization of peptides yield placed under a stream of nitrogen to remove unreacted protonated molecules in which the proton can be coor­ acrolein and acetonitrile, and the remaining moisture dinated by hydrogen bonding along several sites of the was removed by lyophilization. Synthetic thioether peptide backbone [29-35]. Because thioether conjugates conjugates in this study were stored under nitrogen at of N-acety1cysteine have fewer sites for proton coordi­ -30 °C. nation, their fragmentation behavior can be different than larger peptide thioether conjugates. N-Acetylcysteine Conjugates. S-(3-0xopropy1)-N-acetyl­ The principal aim of this study has been to determine L-cysteine (Compound I) was synthesized by dissolv­ relationships between structural features and fragmen­ ing 20 mg of N-acety1cysteine (0.12 mmoles) in one mL tation dynamics in protonated acrolein thioether conju­ of a 1:1 (v.v) acetonitrile:water solution. 20 ILL of gates by using low energy CID tandem mass spectrom­ acrolein (0.30 mmo1) was added by micropipette. 1H etry experiments. This has been accomplished by NMR results are in agreement with spectra published generating energy resolved product ion spectra [23-28] earlier [37, 40] (in d3-acetonitrile): 1.93 ppm (s, acetyl for a series of structurally related peptides that vary in Me, 3 H), 2.67 (tt, -5-CH2--CH 2-CH = 0, C-2, 2 H), 2.77 size and in the number of basic functional groups. (dt, -5-CH2--CH2-CH = 0, C-1, 2 H), 2.95 (dq, Cys-f3, 2 Acrolein thioether conjugates were chosen as model H), 4.55 (dt, Cys-«, 1 H), 6.89 (d, amide, 1 H), 9.65 (t, compounds for Michael-thioether conjugates. Acrolein -5-CH2--CH2--CH = 0,1 H). ESI MS results: m i z 220 is an important industrial chemical [1,361, a metabolite [M + Hl". of the cancer chemotherapeutic agent cyclophospha­ Conversion of 5.2 mg of I (0.024 mmol) to S-(3­ mide, [36-38] and an unwanted side-product of com­ hydroxypropy1)-N-acetyl-L-eysteine (Compound II) was bustion and cigarette smoke [38, 39]. Compounds for accomplished by dissolving I in 2 mL 95% ethanol! this study are acrolein conjugates of N-acetyl-L-cys­ water followed by addition of 1 mg NaBH4• The mix­ teine, glutathione, and N-acetylglutathione. Derivatives ture was sonicated for 20 minutes in an ultrasound of the aldehyde groups were prepared from these bath. An additional 1 mg of NaBH4 was added and the conjugates to assess whether common derivatization of mixture sonicated for an additional 10 min. Unreacted the aldehyde group would alter fragmentation behav­ NaBH4 was quenched by dropwise addition of 0.1 N ior. Structures of thioether conjugates used in this study HCI (until gas evolution stopped, pH 4.0). Purification are displayed in Figure 1. of II was performed by using a Sephadex QAE-A-50 J Am Soc Mass Spectrom 1997, 8,727-736 FRAGMENTATION OF PROTONATED ACROLEIN THIOETHER CONJUGATES 729 anion exchange column. The column was pretreated 2 H), 2.86 (m, Cys-{3, 2 H), 3.64, (t, - 5-CH2- CH2-eH 2­ with successive washes with absolute ethanol and wa­ OH, C-3, 2 H), 3.90 (t, Glu-a, 1 H), 3.98 (s, Gly-a, 2 H), ter. The quenched reaction product was loaded on the 4.56 (m, Cys-o, 1 H). EST MS: m / z 366 [M + H] + . anion exchange column and the column was washed To synthesize the oxime S-O-N-hydroxyiminopro­ with water to elute residual sodium ion.