Anal. Chem. 1990, 62,1827-1836
1827
on the transmission of ionic species. So far no direct experimental evidence has been found for this effect, although time-resolved measurements support some of our predictions. Different masses are transmitted a t different phase angles, and the critical interval of transmitting phase angles is larger for higher masses, indicating a mass discrimination toward lower masses. Modifications of the electric circuitry of the rf spark ion source that reduce the residual electric field oscillations during a spark discharge should lead to better and more stable ion transmission. In the view of new techniques (e.g., GDMS) this can contribute to make SSMS more competitive as an analytical instrument.
try; Adams. F.. Gijbels, R.. Van Grieken, R. E&.; Wiley: New York, 1988 pp 17-84. (4) Plyutto. A. Zh. Eksp. Teor. Fiz. 1860,39, 1589. ( 5 ) Gurevich, A.; Pariiskaya. L.; Pbevskii, L. Zh. €&sp. Teor. Fiz. 1965. 49, 647; 1968. 5 4 , 891; 1972,63, 516. (6) Franzen, J. Z . Naturforsch. 1963, M A , 410. (7) bmendk.G.;Derzhiev, V.; Surkov, Y.; Ivanova, V.; Grechishnlkov, A. IN. J . Mess Spectrom. Ion Phys. 1981, 3 7 , 331. (8) Vertes, A.; Juhasr, P.; De Wolf, M.; Gijbels, R. Scanning Microsc. 1968. 2 . 1853. (9) Vertes, A.;Juhasz. P.; De Wolf, M.; Gljbels, R. Int. J . Mess Spec*Om. Ion Processes 1989. 9 4 , 83. (10) Dahl, D.; Delmore, J. SIMIONK I P S 2 Users Manusi Versbn 4 . 0 , E C G CS-7233 Rev. 2, 1988. (11) Van Puymbroeck, J.; Gijbels, R.; Viczian, M.; Comides. I. Int. J . Mess Spectrom. Ion Processes 1984,56, 269.
LITERATURE CITED
RECEIVEDfor review February 12, 1990. Accepted May 11,
(I) Swenters, K. Ph.D. Thesis, University of Antwerp, 1988. (2) van Straaten, M.; Gijbels, R.; Meichers, F.; Beske, H.; Swenters, K. Int. J . Mess Spctrom. Ion Processes 1989, 93, 125. (3) Ramendik, G.; Verlinden, J.; Gijbels, R. In Inorganic Mess Spectrome-
1990. Mark van Straaten is indebted to the Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL) for financial support.
Screening Strategy for the Detection of Derivatized Glutathione Conjugates by Tandem Mass Spectrometry Paul G. Pearson, William N. Howald, and Sidney D. Nelson* Department of Medicinal Chemistry, BG-20, School of Pharmacy, University of Washington, Seattle, Washington 98195 A series of glutathione (GSH) conjugates have been analyzed directly by fast atom bombardment tandem mass spectrometry (MS-MS), either in their native forms or following conversion to N-[(benzoyloxy)carbonyi] dimethyl ester derivatives. Upon cdiislon-induced dissociation (CID), structurally informative daughter ions were observed that were diagnostic for the glycine and glutamate residues and characteristic of the chemical nature of the conjugated xenoMotk moiety. For each of the derivatized GSH conlugates examined, the most intense daughter ion arose by the elkninatbn of glycine methyl ester (-89 u), which contrasted with the elimination of glutamate (-129 u) as the major CID fragmentation of native GSH conlugates. The characteristic change in the fragmentation upon derivatization was exploited to develop a classspecific screening strategy for the detection of GSH conlugates by constant neutral loss scanning for the elimination of giyclne methyl ester (-89 u). The analytical utility of this approach was established by identifying three GSH conjugates derlved metabolkaiiy from 1,2-dibr~3-chioropropane and by Characterizing a total of six derivatized GSH conjugates derived from xenobiotics of diverse chemical structure. The converslon of GSH conjugates to their N-[(benzoyioxy)carbonyl] dimethyl ester derivatives conferred excellent chromatographic properties, induced characteristic MS-MS fragmentations, and offered a (53 f 13)-foid increase in abM e Sensnivtty when compared with native GSH conjugates. For relatively hlgh-mass ( 1OOO-u) GSH conjugates, a gain in sensitivity could be achieved by the optimization of lowenergy collision regimens for neutral loss scanning.
-
INTRODUCTION The endogenous tripeptide glutathione (7-glutamylcysteinylglycine; GSH) exists at millimolar concentrations in mammalian systems, and in its reduced form, conjugation with
* Author to whom correspondence should be addressed. 0003-2700/90/0362-1827$02.50/0
GSH serves an important physiological role in the detoxication of reactive electrophilic species (1-4). Typically these reactive species, which are generated metabolically from drugs or other xenobiotics, are short-lived and have been implicated in various xenobiotic-induced toxicities. More recently, however, a number of distinct roles for GSH in the bioactivation of foreign compounds have been established (5); for example, conjugation with GSH represents an obligatory initial event in the bioactivation of dibromomethane (6) and hexachlorobutadiene (7) to reactive intermediates. As a consequence, therefore, analytical strategies to identify GSH conjugates in biological matrices are important, as the structural information obtained affords a unique insight into the chemical nature of the transient electrophilic species from which the GSH conjugate is derived. A variety of mass spectral techniques have been applied to the structural characterization of GSH conjugates of foreign compounds, including electron impact, chemical ionization (B), and desorption chemical ionization (9). However, the thermal lability and the polarity of the GSH conjugates have limited the success of these techniques to only a few examples within this important class of phase I1 drug metabolites. In recent years, the analysis of GSH conjugates has been facilitated by the application of “soft ionization” techniques, including field desorption (10-12), fast atom bombardment (FAB), liquid secondary ion mass spectrometry (LSIMS) (13-20),and liquid chromatography (LC)/thermospray mass spectrometry (21-25). A characteristic of these techniques is the ability to derive molecular weight data from abundant MH+ or M Na+ ions of the analytes, but in general the mass spectra thus obtained are deficient in structurally informative fragment ions. Moreover, fragment ions that may be present at low intensities are often obscured by background ions derived from the matrix or by contaminants of biological origin. These limitations of FAB or LSIMS may be overcome by the application of collision-induced dissociation (CID) methods, employed in conjunction with tandem mass spectrometry (MS-MS) to provide increased selectivity and controlled fragmentation to a chosen analyte (17-19,26,27).A
+
0 1990 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
number of workers have reported daughter ion (MS-MS) spectra of GSH conjugates of acetaminophen obtained by (i) linked scanning and CID in the first field-free region of a double-focusing mass spectrometer (8),(ii) low-energy decomposition (electronvolts in the laboratory frame of reference) in the quadrupole collsion region of a hybrid (BEqQ geometry) tandem mass spectrometer (In,(iii) FAB/MS-MS analysis with a triple-quadrupole instrument (28),and (iv) FAB/MS-MS analysis by mass ion kinetic energy spectrometry scans on a triple-sector mass spectrometer of EBE geometry (18). In addition, daughter ion spectra of GSH conjugates of styrene and styrene oxide have been recorded on four-sector instruments (26,27,29).In each instance, satisfactory mass spectral analysis was preceded by the chromatographic purification of biologically derived GSH conjugates. A complementary analytical approach that utilizes N,Oalkoxycarbonyl or N,O-[ (arylalkoxy)carbonyl] derivatives has been employed to confer excellent chromatographic properties on a variety of glutathione and related conjugates, thus facilitating the purification from biological matrices prior to LSIMS and FAB/MS analysis (19,20, 30). In preliminary reports, we have described the FAB/MS-MS properties of S-(N-methylcarbamoyl)glutathione,a metabolite of Nmethylformamide (19)and methyl isocyanate (31),analyzed as an N-[(benzoyloxy)carbonyl] dimethyl ester derivative. An important observation in these studies was a marked directing effect of the N-[(benzoyloxy)carbonyl] group upon CID fragmentation pathways. In contrast to native S-(Nmethylcarbamoyl)glutathione, which decomposed primarily by the elimination of glutamate as a neutral fragment (-129 u), the N-[(benzyloxy)carbonyl] dimethyl ester derivative underwent a major transition with the expulsion of a neutral fragment corresponding to the elements of glycine methyl ester (-89 u). A knowledge of MS-MS fragmentation processes has served in the design of strategies for the rapid identification, or “metabolic mapping”, of drug metabolites by MS-MS techniques (28, 32, 33). General screening strategies for specific classes of metabolite include parent ion scanning for the qualitative and quantitative analysis of sulfate metabolites, i.e., precursors of m / z 97 (28,34), and constant neutral loss scanning (-176 u) to detect glucuronic acid conjugates of drugs (28). In a similar manner, specific screening strategies centered upon the CID fragmentation of the intact parent drug have been exploited for the recognition of metabolites of nicotine (28)and zonisamide (33). The characteristic fragmentation of underivatized GSH conjugates involving the elimination of glutamate (-129 u) has been exploited for the detection of the GSH conjugate of acetaminophen by constant neutral loss scanning ( 1 7 ) . In contrast, the directing effect exerted upon CID fragmentation by the introduction of an N-[(benzyloxy)carbonyl] functionality facilitates a novel screening strategy based upon constant neutral scanning for the loss of glycine methyl ester (-89 u). The objectives of the present investigation were to exploit the excellent chromatographic properties conferred upon GSH conjugates by a simple aqueous-phase derivatization procedure and thus expedite their isolation from biological matrices. Metabolically derived GSH conjugates thus isolated were subjected to FAB/MS-MS analysis with constant neutral loss scanning for the loss of glycine methyl ester (-89 u) to identify GSH conjugates of unknown structure. As model compounds for this investigation, authentic standards of GSH conjugate metabolites of acetaminophen, trichloroethene, and 1,2-dibromo-3-chloropropane (DBCP) were selected in order to establish common CID fragmentation pathways for a series of conjugates derived from xenobiotics of diverse chemical structure. Furthermore, to realistically evaluate the analytical utility of the proposed screening approach, GSH conjugates
of DBCP generated metabolically in biological matrices were derivatized, isolated, and identified by FAB/MS-MS. In pursuit of these objectives, experiments were designed, therefore, to characterize GSH conjugates of diverse chemical structure isolated from a complex biological milieu and to establish appropriate MS-MS collision regimes for the (i) optimal detection of derivatized GSH conjugates by constant neutral loss scanning MS-MS procedures and (ii) the generation of diagnostic daughter ion fragments to aid in the characterization of GSH conjugates containing a single glutathione moiety and more complex GSH conjugates with two glutathione moieties.
EXPERIMENTAL SECTION Chemicals. Benzyl chloroformate was purchased from Aldrich Chemical Co. (Milwaukee, WI). Glutathione and oxidized glutathione (GSSG) were obtained from Sigma Chemical Co. (St. Louis, MO). [U-14C]DBCP(23.6 mCi mmol-‘; 97% radiochemically pure) was obtained as a gift from the Shell Development Co. S-(1,2-dichlorovinyl)glutathione(DCVG),prepared according to the method of Mckinney and Beister (3.9,was a gift from Dr. E. A. Lock (IC1 CTL Cheshire, England). 3-(GlutathionSyl)acetaminophen (APAP-SG) was prepared by the reaction of N-acetyl-p-benzoquinoneimine with glutathione and was purified by chromatography on Sephadex LH-20 (Pharmacia, Inc., Piscataway, NJ) as previously described (30, 36). S-(2,3-Dihydroxypropy1)glutathione (DHPG), S-(3-chloro-2-hydroxypropy1)glutathione (CHPG), and 1,3-bis(glutathion-S-y1)propan-2-01 (HPBG) were prepared by the reaction of glycidol, epichlorohydrin, and epibromohydrin, respectively, with glutathione and were purified as N - [ (benzyloxy)carbonyl]derivatives as previously reported (37). DCVG, GSSG, APAP-SG, DHPG, CHPG, and HPBG were converted to N-[(benzyloxy)carbonyl] dimethyl ester derivatives by successive treatments with benzyl chloroformate and/or anhydrous methanolic hydrogen chloride, prior to mass spectrometric analysis (19). When required, the N- [ (benzyloxy)carbonyl] functionality was effectively removed from DHPG, CHPG, and HPBG by hydrolysis in 5 N HC1 for 2 h at ambient temperature. [ 1,1,2,3,3-2H5]-2,3-Dibromochloropropane (D5-DBCP)was prepared as previously described (38); the isotopic composition was determined to be 16% 2H4and 84% 2H5based on the M - Br ions at m/z 155-165 observed upon electron impact mass spectrometric analysis. Biological Experiments. For metabolic experiments to detect GSH conjugate metabolites of DBCP, male Sprague-Dawley rats (200-225 g; Charles River Laboratories, Inc., Wilmington, MA) were anesthetized with sodium pentobarbital and their bile flow was exteriorized via a polyethylene cannula (PE-10). DBCP (80 mg kg’) or the perdeutero analogue (D,-DBCP) was administered with a radiotracer of [U-14C]DBCP(1000 dpm nmol-I), and bile was collected for a 6-h period. In other studies, the coadministration of equimolar doses of the parent drug and a stable isotopically labeled variant has aided in metabolite identification by the “twin-ion technique” (17). In the present investigation, the separate administration of DBCP and D,-DBCP was required to avoid perturbation of the “twin-ion” clusters by deuterium kinetic isotope effects upon the cytochrome P-450 mediated oxidation of DBCP to reactive metabolites (39). High-Performance LC Purification of Glutathione Conjugates. The purification of N-[(benzyloxy)carbonyl]glutathione conjugates of DBCP or D5-DBCP isolated from the bile of rats was performed on a system equipped with two Waters M6000A pumps, a Waters 440 UV detector (A = 254 nm), and a Waters 680 high-performance LC gradient controller (Waters Associates, Milford, MA). The separation of GSH conjugates was achieved on an reverse-phase Altex 5 pM Ultrasphere ODS column (25 cm X 10-mm i.d., Rainin Instruments, Berkley, CA). The elution mobile phase consisted of a linear 30-min gradient from 30% to 95% methanol in water, with 1% acetic acid throughout, followed by isocratic elution at 95% methanol for an additional 10 min. The flow rate was held at 3 mL m i d , and fractions of the column effluent were collected at 30-s intervals with a Pharmacia Frac-100 fraction collector (Pharmacia). Radioactive GSH conjugate metabolites of DBCP were visualized by liquid scintillation counting of aliquots (500fiL)of the high-performance LC fractions.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
The major radioactive fractions eluting from the high-performance LC column at 11-12, 15-16, and 19.5-20.5 min were isolated, converted to their N- [ (benzyloxy)carbonyl] dimethyl ester derivatives, and subjected to FAB/MS and FAB/MS-MS analysis. Mass Spectrometry. FAB/MS spectra were recorded on a VG 70-SEQ hybrid tandem mass spectrometer of EBqQ geometry (VG Analytical Ltd., Manchester, U.K.), equipped with a saddle-field fast atom gun (Ion Tech Ltd., Teddington, Middlesex, U.K.) and a VG 11/250 data system. The samples (1-5 wg) were dissolved in methano1:l N HC1 (1:l; 50 wL), and aliquots (1 WL) were added to a glycero1:thioglycerol:methanol (1:l:l)matrix on a FAB target. Sample introduction was performed with a conventional FAB probe, and ionization was achieved following bombardment with a primary beam of xenon (8 ke V). Conventional FAB/MS spectra were recorded via the data system at an accelerating voltage of 8 kV and a nominal mass resolution of M/AM = 2000 (10% valley). Daughter ion spectra (MS-MS) of the MH+ ions of candidate metabolites of DBCP were obtained by CID in the first (radio frequency (rf) only) quadrupole. The collision energies were controlled by floating the rf-only quadrupole at potentials from 5 to 100 V below the accelerating voltage (8 kV). The collision energies,therefore, varied between 5 and 100 eV in the laboratory frame of reference. The parent MHI ions of the analytes were selected by the adjustment of the magnetic field strength of the magnet (B), and daughter ion spectra were recorded by scanning the quadrupole mass analyzer (Q) from m/z 50 to 1200 over a period of 10 s. The spectra were recorded in the MCA (multichannel analyzer) mode; 5-10 scans were summated, centroids were assigned, and the peaks were subsequently mass measured by the data system. Constant neutral loss scanning to screen for biliary GSH conjugate metabolites of DBCP was performed by a linked scan of the magnet (B) and quadrupole mass analyzer (Q) at a constant mass offset of 89 u, corresponding to the elimination of glycine methyl ester. For neutral loss scanning, optimum collision parameters were established at collision energies of 35 eV with argon as a target gas (3.6 X 10"' Torr). Daughter Ion Spectra as a Function of Derivatization, Collision Cell Gas Pressure, and Collision Energy. The CID methyl daughter ion spectra of the GSSG-N-[(benzyloxy)carbonyl] ester derivative were monitored as a function of collision cell gas pressure and collision energy to establish appropriate experimental parameters for either constant neutral loss scanning or structural elucidation. Argon was employed as a collision gas in the quadrupole collision region at pressures of either 2 X lo4 Torr or 8 X lo4 Torr measured in the quadrupole analyzer housing. Torr, These pressures correspond to 3.6 X lo4 Torr or 1.4 X respectively, in the quadrupole collision region based upon gas conductance calculations relevant to the VG 70-SEQ instrument. Daughter ion spectra were recorded by CID on MH+ = 937; the collision energies were varied by increments of 5 eV between 0 and 50 eV and subsequently by increments of 10 eV between 50 and 100 eV. For each of the two gas cell pressures and for each increment of collision energy, a daughter ion spectrum was recorded; in each case, 20 scans of the quadrupole mass analyzer (50-1200 u) were accumulated in the MCA mode. The effect of the collision cell gas pressures upon the daughter ion spectra was expressed by plotting the percent daughter ion current for the ions at m/z 159,468, and 848 as a function of the collision energy for each of the gas pressures chosen. The influence of the derivative formation upon the field of the appropriate fragment ion for the detection by neutral loss scanning was determined by monitoring the relative conversion of the MH+ ion from MS-1 to -129 u or -89 u in MS-2 for the native and derivatized GSH conjugates, respectively. Specific conversions monitored for derivatized GSH conjugates were CHPG m / z 562 (MH+in MS-1) to m / z 473 (ion a in MS-2) and GSSG m / z 937 (MH+ in MS-1) to m / z 848 (ion a in MS-2). For native GSH conjugates, transitions involving the loss and glutamic acid were monitored: CHPG m / z 402 (MH+ in MS-1) to m / z 273 (ion e in MS-2) and GSSG m / z 613 (MH+ in MS-1) to m / z 484 (ion e in MS-2). For each set of determinations, the transmission of the selected MH+ from MS-1 to MS-2 was measured as a voltage with the instrument oscilloscope and subsequently corrected for differences in multiplier gain from MS-1 to MS-2. The intensity of either ion a or ion e, relative to the MH+ ion in MS-2, was
+ //"HZ
I XS/C" g
1829
+
CH
I1
XdCH h
fragmentation of derivatized GSH conjugates upon low-energy collision-induced dissociation.
Figure 1. FAB/MS-MS
determined by 10 scans of the quadrupole mass analyzer, which were accumulated in the MCA mode.
RESULTS AND DISCUSSION In order to establish the general utility of MS-MS approaches to the characterization of GSH conjugates and, more specifically, the value of constant neutral loss scanning for the detection of GSH conjugates, a series of GSH conjugates that are derived metabolically from foreign compounds of toxicological significance have been subjected to FAB/MS and FAB/MS-MS analysis, either in their native forms or as N - [(benzyloxy)carbonyl] dimethyl ester derivatives. Each of the chosen analytes is representative of a distinct class of GSH conjugate from the perspective of metabolic origin, toxicokinetics, and, more significantly, the chemical nature of their thioether linkage with the cysteinyl thiol of GSH. Conventional FAB/MS examination of each of the derivatized conjugates revealed intense protonated molecular ion (MH+) species; in general, however, the spectra thus obtained were devoid of structurally informative fragment ions. When the MH+ ion of each of the analytes was selected and subjected to CID (40-50 eV and 3.5 X Torr), a series of abundant structurally informative daughter ions were observed; the daughter ion spectra are tabulated in Table I, and the origins of the individual daughter ions are described according to standard nomenclature initially proposed by Gaskell and co-workers (17) and subsequently adopted in our laboratory (Figure 1) (19, 31). The daughter ion spectra of the.derivatized GSH conjugates may, in general, be rationalized by four classes of fragmentation, as exemplified by the daughter ion spectra of 36C12 (MH+ = 564) and 37C12(MH+ = 568) pseudomolecular ions of the DCVG N - [(benzyloxy)carbonyl] dimethyl ester derivative (Figure 2, A and B, respectively). The primary route of fragmentation occurred by the elimination of the elements of glycine methyl ester (-89 u) from the MH+ species at m/z 564 or mlz 568 to afford an intense ion a at m / z 475 (Figure 2A) or m / z 479 (Figure 2B), respectively. For each of the derivatized GSH conjugates examined, the elimination of glycine methyl ester was observed to be the primary mode of decomposition. In contrast, the elimination of glycine (-75 u) was a structurally informative mode of fragmentation for native GSH conjugates, but in each example, the pathway to ion a was quantitatively less important than decomposition by the elimination of glutamic acid (-129 u) to form ion e. In accord with earlier findings (17,19,28), this second mode of fragmentation involving the elimination of glutamate (-129 u) was the predominant CID decomposition pathway for underivatized GSH conjugates (Table I). Conversely, for each of the derivatized GSH conjugates, the elimination of the derivatized glutamyl moiety (-277 u) to afford the protonated cysteinylglycine methyl ester derivative (ion e) was a struc-
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
Table I. Daughter Ion Spectra of [M Glutathione Conjugates" parent drug
+ H]+Ions from the Native or N-[(Benzyloxy)carbonyl] Dimethyl Ester Derivatives of Synthetic [M + H]+
substituent X
[M + H]+ H20
[M + H]+ COP
[M + H]+ PhCHzOH
500 (9) 518 (11) 520 (8) 520 (17) 524 (12) 709 (15)
436* (10) 454 (7) 456 (6) 430 (7)
Derivatized Glutathione Conjugates
37C1CIC=CH37C1 acetaminophen
37C1C=CH37C1 APAP-SG
BrCH2CHBrCH2Cl BrCH2CHBrCHIS6CI BrCH;CHBrCHiS7C1 36CIC1C=CHBbCI 37CIClC=CH37CI acetaminophen
CHgCHOHCH2OH CHzCHOHCHzS6C1 CH;CHOHCH;3TCI WC=CH"Cl 37CIC=CH3'C1 0-CBZ-APAP-SGh
544 562 564 564 568 753
526 (3) 544 (4) 546 (3) 546 (4) 548 (2) 735 (3)
645 (5)
Native Glutathione Conjugates 382 400 402 402 406 457
364 (8) 384 (2) 388 (5)
Given as m/z values. Numbers in parentheses are abundances of characteristic daughter ions expressed relative to the most intense daughter ion (see Figure 1 for identities of ions a-k). CID was performed in the quadrupole collision region of a VG7O-SEQ hybrid tandem instrument of EBqQ geometry. Argon was used as a collision gas at a pressure of 3.5 X lo-' Torr and collision energies were 40-50 eV. *Isobaric ions. - COP dMH+- 134 - HSX. 'e - X H. fMH+ - 363. Be - HSX. hC7H7+. i a - 134. j a - PhCHzOH. k c - HzO. ' e - HzNCOOH. "'a - HSX. "0-CBZ = 0-[(benzyloxy)carbonyl]. "182 - 42. p e - 42.
+
turally informative mode of fragmentation but was quantitatively leas significant than the corresponding fragmentation (-129 u) in the native GSH conjugates. The third class of daughter ions arose via cleavages adjacent to the cysteinyl thiol. In the example of DCVG, the further fragmentation of ion e afforded an ion at m f z 159 (Figure 2, A and B). This ion, which arises by the loss of the conjugated xenobiotic moiety accompanied by SH, involves the expulsion of the parent drug molecule, and when this fragmentation is operative, a characteristic daughter ion is observed in the CID spectra of derivatized conjugates, the mass of which is independent of the chemical nature of the conjugated xenobiotic moiety. The final class of fragmentation occurred via the elimination of small neutral fragments characteristic of the functional groups (e.g., COz, 44 u; PhCH,OH, 134 u; H20, 18 u); these ions noted as "other ions" in Table I were generally not observed upon collision activation of native GSH conjugates and have been ascribed to processes involving the functional groups (19). Upon collision activation, therefore, characteristic daughter ions arose that provided structural information concerning the presence of the glutamyl and glycyl residues and the nature of the conjugated drug moiety. In addition, specific qualitative and quantitative changes in fragmentation were observed that were dependent upon the chemical nature of the conjugated xenobiotic moiety. These changes were, in general, restricted to fragmentation processes occurring adjacent to the cysteinyl thiol and varied from simple quantitative changes in ion intensities to qualitative changes in daughter ion formation (Table I). The quantitative changes were typified by variation in the intensity of the ion a t m / z 159, which arises by the fragmentation of ion e with the elimination of the xenobiotic moiety accompanied by SH. This was a major pathway of fragmentation for simple S-alkyl-substituted GSH conjugates such as DHPG and CHPG and yields ions of approximately 70% of the abundance of the most intense daughter ion in the spectra of each of these derivatized conjugates. It is interesting to note that this intense ion, which is informative of the nature of the conjugated xenobiotic moiety, is not present in the CID spectra of the native GSH conjugates and thus arises due to the induction of a specific fragmentation upon derivatization (Table I). For derivatized conjugates, however, the presence of electron-withdrawing substituents adjacent to the thiol, as for DCVG and also for S-(Nmethylcarbamoy1)glutathione (19),suppressed this pathway of fragmentation. In an analagous manner, the intensities of other ions formed by processes involving cleavage adjacent to the thiol, e.g., m / z 302, are subject to similar variations in
intensity. Qualitative differences in fragmentation induced by the xenobiotic moiety were restricted to APAP-SG, which was the only GSH conjugate examined in which a significant ion d was observed (see Table I). This mode of fragmentation was unchanged between derivatized and native APAP-SG, with the exception of a shift in mass of ion d by 150 u associated with the formation of an N,O-[(aryloxy)carbonyl] dimethyl ester derivative of APAP-SG (30). While the functionalization of the hydroxyl moiety of the 4-hydroxyacetanilide substituent incurred an increase in mass, the derivatized conjugate behaved in a controlled and predictable fashion upon CID analysis. A striking observation upon comparison of the CID daughter ion spectra of derivatized and native GSH was the marked directing influence upon fragmentation induced by derivatization. In each instance, the most intense daughter ion arose via the elimination of methylglycine (89 u; ion a) and was independent of the chemical nature of the conjugated xenobiotic moiety. The quantitative changes in the relative intensities of ion a and ion e observed upon derivatization is due to a directing effect upon fragmentation exerted by the introduction of an N-[(benzyloxy)carbonyl] functionality. The acetylation of the glutamate amino terminal has also been observed to induce a similar directing effect upon fragmentation (In,suggesting that the expulsion of the glutamyl moiety occurs by a charge site directed process with the elimination of pyroglutamate as a stable neutral fragment. In contrast, the formation of monomethyl or dimethyl ester derivatives of S-(N-methylcarbamoy1)glutathionedid not alter the relative yields of ion a and ion e when compared with the native GSH conjugate (39). Neutral Loss Screening for Glutathione Conjugates: Qualitative Aspects. A prerequisite for a screening strategy based upon tandem mass spectrometry is the ability to generate daughter ions in a controlled manner, which are specific to the class of metabolite under consideration and independent of the nature of the conjugated xenobiotic. For each of the conjugates examined, the derivatized GSH component fragmented in a predictable fashion by a neutral loss of glycine methyl ester, a process that was independent of the chemical nature of the conjugated xenobiotic moiety. As a consequence, therefore, the elimination of glycine methyl ester was subsequently exploited to develop a class-specificMS-MS constant neutral loss screening strategy for the detection of GSH conjugates. As model compounds, GSH conjugates derived metabolically from DBCP were identified by this screening procedure. The conventional FAB/MS spectrum displayed in Figure 3A was recorded for a metabolite of DBCP excreted
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
a
C
d
e
f
g
h
1
other ions
k
455 (100) 250 (5) 473 (100) 268 (8) 475 (100) 270 (5) 475 (100) 479 332 (18) 664 (100)
267 (28) 285 (31) 287 (21) 287 (17) 291 (36) 476 (17)
278 (6) 278 (5) 278 (6) 278 (16) 278 (21) 278 (12)
150 (11) 133 (5) 468 (43) 436* (10) 168 (18) 170 (10) 170 (10) 174 (21) 436 (7) 359 (7) 342 (5)
41lC(15) 42gC(6) 43lC(7) 52oC (17) 524c(20) 62OC(25)
307 (24) 325 (15) 327 (20) 327 (14) 331 (13) 382 (54)
253 (100) 271 (81) 273 (100) 273 (38) 277 (39) 328 (100)
130 (29) 130 (7) 130 (5) 130 (25) 130 (27) 130 (8)
150 (65) 168 (100) 170 (93) 170 (100) 174 (100) 225 (11)
218' (15) 177' (33) 199"' (15) 236' (30) 199m (6) 238' (21)
236 (81) 254 (69) 256 (62) 256 (19) 260 (21) 311 (18) 182 (46)
133 (14) 151 (20) 153 (27) 153 (3) 157 (4) 208 (15)
in the bile of rats. This spectrum displays an abundant protonated MH+ ion at m / z 544 accompanied with M + Na+ and M + K+ ions at m / z 566 and 582, respectively, which are consistent with the presence of DHPG as a biliary metabolite of DBCP. The spectrum in Figure 3A is also characterized by abundant ions derived from the matrix or from contaminants of biological origin, which obscure any structurally informative fragment ions that may be present at low abundance. However, when the same sample was reanalyzed in a constant neutral loss experiment in which B and Q were scanned simultaneously but with a constant mass offset of 89 u, the spectrum thus obtained (Figure 3B) displayed an intense ion at m / z 544, with almost total elimination of the intense background peaks that were observed in conventional FAB/MS analysis (Figure 3A). The small peaks indicated at m / z 470,436, and 159 (Figure 3B) are readily rationalized as fragment ions of MH+ = 544 formed in the source. Each of these ions is detected in the neutral loss experiment, as they possess the substructural component of glycine methyl ester. The origin of m / z 159, for example, is illustrated in Figure 3C and Table I. A comparison of the conventional FAB/MS spectrum and the neutral loss spectrum clearly illustrates the selectivity of the MS-MS screening approach and its utility, in this instance, for the rapid identification of the ion at m / z 544 as a GSH conjugate. Structural confirmation was subsequently obtained from a daughter ion spectrum recorded on the putative molecular ion species at m / z 544. Upon collision activation of the MH+ ion at m / z 544, a series of abundant structurally informative daughter ions were observed (Figure 3C); fragment ions arose from the loss of glycine methyl ester (-89 u; ion a) and glutamic acid N-[(benzyloxy)carbonyl]methyl ester (-277 u; ion e). The further fragmentation of ion e adjacent to the cysteinyl thiol to afford the ion at m / z 159 by a loss of 108 u was diagnostic for the 2,3-dihydroxypropyl moiety conjugated to GSH. In common with the CID daughter ion spectrum of DCVG (Figure ZA), further fragment ions of DHPG were observed with the elimination of small neutral fragments characteristic of several molecular species (e.g., COP,44 u; PhCH,OH, 134 u; H20, 18 u). The daughter ion spectrum of the biologically derived DHPG compared favorably with that reported in Table I for an authentic sample of DHPG. The utility of this MS-MS screening aproach was further evaluated by screening additional isolates of biliary metabolites of DBCP. A constant neutral loss of MS-MS spectrum of a biliary metabolite of DBCP is depicted in Figure 4A, in which the spectrum is dominated by an intense ion at m / z 562 accompanied by a T 1 satellite ion at m/z 564, consistent with
1831
302d(17) 191' (5) 302d (35) 302d (20) 302" (6) 302" (10) 302d (6)
181' (44) 199' (15) 2011 (5) 341' (10) 345' (7) 556' (12)
159 (62) 159 (76) 159 (80) 15W (11) 159 (11) 159 (42)
91' 91' 91' 91' 91' 91'
(8) (12) (8) (20)
(16) (11)
1400 (16) 268p (3)
the presence of a GSH conjugate of DBCP containing a single atom of chlorine. A daughter ion spectrum was recorded on the putative MH+ ion a t m / z 562, which displayed a series of daughter ions (Figure 4B) qualitatively similar to those observed for DHPG. Abundant diagnostic ions were observed for the glutamyl (mlz 285; ion e) and glycyl (m/z 473; ion a) components; the further fragmentation of ion e to yield an ion at m / z 159 by the loss of HSCH2CHOHCh296Cl(-126 u) was informative for the nature of the conjugated xenobiotic moiety. The facile identification of DHPG and CHPG as biliary metabolites of DBCP clearly illustrates the value of a neutral loss scanning approach based upon the loss of 89 u to identify simple S-alkyl-substituted GSH conjugates of DBCP. The value of MS-MS approaches to the analysis of GSH conjugates, following chemical derivatization, was further established by the consideration of a mixture of GSH conjugates of biological origin, in which various analytes within the sample contained two moieties of GSH. Conjugation with GSH in which two moieties of GSH become conjugated with the xenobiotic is unusual, but examples have been described for metabolites of hexachlorobutadiene (40) and 3-hydroxyacetanilide (41). The analysis of poly-GSH conjugates by FAB/MS-MS techniques, however, has remained undocumented. In the present example, general principles of fragmentation have been established that facilitated the recognition of poly-GSH conjugates of moderately high mass (- 1000 u) by constant neutral loss scanning techniques. Conventional FAB/MS of a biliary metabolite of DBCP (Figure 5A) revealed the presence of a number of abundant ions within this complex sample; many of these ions were of similar intensity to the molecular ion species that is indicated at m / z 995. Constant neutral loss (-89 u) spectra were subsequently recorded in order to identify molecular ion candidates of putative GSH conjugates derived from DBCP (Figure 5B) or a perdeutero analogue thereof (D5-DBCP;Figure 5C). A comparison of parts A and B of Figure 5 clearly indicates the presence of intense ions at m / z 995,937, and 613, which were evident in the conventional FAB/MS spectrum but at significantly lower abundance than were observed in the constant neutral loss spectra. The isotopically induced shift in the mass of the ion at mlz 995 to m / z 1000 upon analysis of the D5-DBCP metabolite established the presence of a component derived from DBCP, in addition to endogenous GSH-related components at m/z 613 and 937. The structures of the MH+ ions at m / z 937 and 1000 were established from their respective daughter ion spectra as the derivatized forms of GSSG and 1,3-bis(glutathion-S-y1)-[ 1,1,2,3,3-W5]propan-Z-ol
1832
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990 MH*
A
A
5
r
"1 'O0I
438
i'iI 'r
0
1
367
I
200
100
5r
I
MH'
400
300
500
600
70C
L,
BW
B loo
MH'
1
T %
50.
300
200
0100
4w
600
500
rnz
Figure 2. (A) Daughter ion (MS-MS) spectrum obtained by collision activation of the 35C12satellite of the MH' ion (at m l z 564) of derivatized DCVG. The structure of the analyte and the proposed origins of the daughter ions are as indicated. Collision-induced dissociation was performed in the first (rf-only) quadrupole (4) of a VG 70-SEQ hybrid tandem mass spectrometer (EBqQ geometry) with argon as a collision gas (3.6 X lo-' Torr in the coHision region) and collision energy of 40 eV. (B) Daughter ion (MS-MS) spectrum obtained by collision activation of the 37C12satellite of the MH' ion (at r n l z 568)of derivatized DCVG. The structure of the analyte and the proposed origins of the daughter ions are as indicated; a shift in mass of 4 u between certain ions in spectra A and B is diagnostic for fragment ions arising with retention of the dichlorovinyl moiety.
or a regioisomer thereof (Figure 6, A and B). The daughter ion spectra for both conjugates were rationalized by two modes of fragmentation that were in accord with the fragmentation scheme proposed for simple S-alkyl-substituted GSH conjugates (Figure 1)as illustrated by the consideration of the CID spectrum of m/z loo0 (Figure 6A): (i) Structurally informative ions arose from neutral losses ascribed to the glycine and glutamate moieties or from the loss of neutral species characteristic of the functionalized carboxylate or amino groups. The predominant route of fragmentation involved the elimination of glycine methyl ester to ion a at m/z 911. The presence of an additional glycine and, by inference, a second GSH component was established by a second loss of methylglycine to yield ion a' at m / z 822. The observation of an ion at m / z 723 was informative for the loss of the glutamate component (-277 u); however, this was quantitatively less important than was previously established for S-alkyl-sub-
C 544
100-
%
50.
I
91
267
357
0 100
2w
300
400
Figure 3. (A) Conventional FABlMS spectrum of a derivatized extract of bile collected from a rat administered DBCP. The MH' and M iNa' ions for DHPG are evident at m l z 544 and 566, respectively. (B) MS-MS spectrum obtained by constant neutral loss scanning for the loss of methyglycine (-89 u) from the same sample used to acquire the spectrum shown In A. cdlisioninduceddissociation was performed in the first (rfonly)quadrupde (4) of a VG 70-SEQ hybrid tandem mass spectrometer (EBqQ geometry) with argon as a collision gas (3.6 X lo-' Torr in the collision region) and collision energy of 40 eV. The presence of an intense ion at m l z 544 illustrates the selectivity of this technique. (C) Daughter ion (MS-MS) spectrum obtained by colllsbn activation of the MH' ion (at m l z 544) of derivatized DHPG isolated from the bile of a rat administered DBCP. The structure of the metabolie and the proposed origins of the daughter ions are as indicated.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
1833
A 100.
937
i"'
m
a 68
I
I'
I
I I
l . i 11w
700
.x
3.0
"id
115
800
518
800
1200
1wO
mh
m/z AH+
Figure 4. (A) MS-MS spectrum obtained by constant neutral loss scanning for the loss of methylglycine (-89 u) from a biliary metabolite of DBCP. Prominent ions are apparent at r n l z 562 and 564, suggasting the p~esenceof an t M ion of a gbtathbne conjugate of WP containing one chlorine atom. (B) Daughter Ion (MS-MS) spectrum obtained by collision activation of the 35Cisatellite of the MH+ ion (at r n l z 562) detected in A by constant neutral loss scanning. The structure of the metabolite and the proposed origins of the daughter ions are as indicated. Collision-induced dissociation was performed as described in Figure 2. stituted mono-GSH conjugates. (ii) The second class of fragmentation gave rise to a series o f low-mass ions previously recognized for DHPG (Figure 2C and Table I); these were attributed t o processes involving the fragmentation of the GSH moiety. Fragmentation adjacent t o either o f the two cysteinyl thiols afforded ion iand ion k as observed for DHPG (Table I). These ions and the observation of a complementary ion a t m/z 565 (Figure 6A) were informative o f the structure and deuterium enrichment of the propan-2-01thioether linkage between the two GSH components. Similar considerations were applied to assign the daughter ion spectrum of m / z 937 t o derivatized GSSG (Figure 6B). N e u t r a l Loss S c r e e n i n g for G l u t a t h i o n e Conjugates: Q u a n t i t a t i v e Aspects. An MS-MS screening approach for the detection o f GSH conjugates i s subject t o a number o f variables. These include the yield o f the parent MH+i o n desorbed f r o m the matrix, t h e experimental parameters for the CID fragmentation of the MH' i o n via the neutral loss monitored in the screening process, and the subsequent transmission o f the required daughter i o n in the second mass spectrometer. Moreover, empirical observations in our lab-
'
O
r
I
m/z Flgure 5. (A) Conventional FABlMS spectrum of HPBG isolated from the bile of a rat administered DBCP. The MH+ ion of HPBG (at m l z 995) and structurally informative fragment ions arising from the loss of the glutamate (ion e at m l z 723) or glycine (ion a at rnlz 911) moieties are apparent. (B) MS-MS spectrum obtained by constant neutral loss scanning for the loss of methyiglycine (-89 u), performed on the same sample employed in A. Prominent ions are apparent at m l z 937 and 995, suggesting the presence of multiple components containing the substructural elements of glutathione. Collision-induced dissociation was performed as described in Figure 38. (C) MS-MS spectrum obtained by constant neutral loss scanning for the loss of methylglycine (-89 u), performed on the same metabolite analyzed in B but derived from D5DBCP. Prominent ions are apparent at m l z 937 and 1000, suggesting the presence of a GSH conjugate of DBCP at m l z 1000 and an endogenous biliary constituent at m l z 937.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
1834
YO
20
0
40
60
80
100
Collision Energy (eV) rnh
B
a 848
I
loo-
1
80
2
-
Argon 3.6 I iO%rr
60
-
Oh
I
1
I
40937
i
i
i
mm 0
0
m z
Figure 8. (A) Daughter ion (MS-MS) spectrum obtained by collision activation of the MH+ ion (at mlz 1000) of derivatized metabolite of DBCP detected in rat ble by constant neutral loss scannlng (seeFigure 5C). The structure of the metabolite and the proposed origins of the major daughter ions are as indicated. Collision-induced dissociation was performed in the first (rf-only) quadrupole (9) of a VG 70-SEQ hybrid tandem mass spectrometer (EBqQ geometry) with argon as a collision gas (1.4 X Torr) and collision energy of 35 eV. (B) Daughter ion (MS-MS) spectrum obtained by collision activation of the MH+ ion (at mlz 937) of derivatlzed GSSG detected in rat bile by constant neutral loss scanning (see Figure 5C). The structure of the metabolite and the proposed origins of the major daughter ions are as indicated.
oratory suggested that for higher mass GSH conjugates, the low-energy collision parameters required for optimum neutral loss screening were inappropriate for structural elucidation. As a consequence, therefore, experimental variables for the detection of GSH conjugates by neutral loss screening were examined, and the effect of chemical derivatization upon each of these was established. The chemical derivatization approach employed in this report results in a quantitative change in the yield of daughter ions upon collision activation of GSH conjugates and facilitates a screening strategy based on the loss of 89 u (ion a), compared to a loss of 129 u (ion e) for native GSH conjugates. The inherent sensitivity of either screening strategy is dependent on the relative conversion (transmission) of the parent to either ion a or ion e upon CID. This parameter is largely dependent on two independent variables, viz., collision cell gas pressure and collision energy. Empirical observations in our laboratory suggested that for higher mass GSH conjugates collision cell
20
40
60
80
I00
Collision Energy (eV)
Figure 7. Ion intensity diagram illustrating the relative intensities of ions at mlz 159, 468, and 848 obtained upon collision acthratlon of the parent MH+ ion (at mlz 937) of the GSSG N-[(benzylOxy)carbonyI] derivative. Argon was used at a pressure of 1.4 X 10" Torr, and the collision energy was incremented as detailed in the Experimental Section. (B) Ion intensity diagram illustratingthe relative intensities of ions at mlz 159, 468, and 848 obtained upon collision activation of the parent MH+ ion (at mlz 937) of the GSSG N-[(benzybxy)carbonyI] derivative. Argon was used at a pressure of 3.6 X lo-' Torr, and the collision energy was varied as indicated in A.
pressures of 1.4 X Torr were required for structural elucidation. Optimal conditions for neutral loss scanning, however, required significantly lower gas cell pressures (3.6 X lo4 Torr). The effect of these two collision cell pressures on the relative yields of ions at mlz 159, 468, and 848 (ion a), generated by CID of the MH+ ion of GSSG N-[(benzyloxy)carbonyl] methyl ester derivative at various increments of collision energy between 5 and 100 eV (laboratory frame of reference), are depicted in Figure 7, A and B. These ion intensity diagrams illustrate graphically that at 1.4 X lV3Torr, significant yields of lower mass ions ( m / z 159 and 468) may be obtained with increasing collision energy but at the expense of the reduction in the intensity of mlz 848 (ion a) and the parent MH+ = 937 (data not shown). From these considerations, optimum conditions of 35-40 eV and 1.4 X Torr were established for structural elucidation of higher mass GSH conjugates. Optimum collision regimes for neutral loss scanning were similarly established. At lower gas cell pressures (3.6 X Torr), little relative attenuation of ion a was observed with increasing collision energy, and optimum conditions for constant neutral loss scanning (transmission of ion
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
a into MS-2) were established at 40 eV and 3.6 X loa Torr. The two ion intensity diagrams illustrate the relative yields of structurally significant daughter ions observed at each of the c h w n collision cell pressures. However, the absolute yield of ion a was also subject to variation with gas cell pressure. The conversion of the MH+ ion at 937 (MS-1) to ion a (MS-2) was 0.52% f 0.07% under optimum conditions for neutral loss scanning. In contrast, the yield of ion a was 0.24% f 0.08% under conditions employed for structural elucidation. These observations indicate that under the optimized conditions for neutral loss screening, a 108% increase in the absolute intensity of ion a was observed for GSH conjugates of higher mass when compared to the conditions required for the generation of significant yields of lower mass daughter ions. In order to establish the relative merits of neutral loss scanning for the elimination of 129 u (ion e) from native conjugates, compared with the loss of 89 u (ion a) for derivatized conjugates, the relative yields of these ions from CHPG or GSSG in the native or derivatized forms was established under the optimal neutral loss conditions. In the case of GSSG, the conversion of the parent ion in MS-1 (MH+ = 613) to ion (m/z 484) in MS-2 was 0.50% f 0.97%, which compares closely with 0.52% f 0.07% observed for the conversion of derivatized GSSG (MH+ = 937) to ion a ( m / z 848). In a similar fashion, for CHPG, the conversion of parent ion in MS-1 (MH+ = 400) to ion e ( m / z 271) in MS-2 was 1.26% f 0.19%, which compared closely with the value observed for the conversion of the N-[(benzyloxy)carbonyl] derivative of CHPG (MH+ = 562) to ion a ( m / z = 473; 1.29% f 0.13%). For both CHPG and HPBG, the similarities between the yields of ion a and ion e upon collision activation of derivatized and native GSH conjugates, respectively, indicates that neutral loss screening approach based upon the loss of glutamate or glycine methyl ester compares favorably with respect to transmission considerations. The relative sensitivity of small peptides in FAB/MS or LSIMS analysis may be related to a relative hydrophilicity/hydrophobicity index (AG),in which small hydrophilic peptides display poor sensitivity (42). GSSG is a hydrophilic peptide with a strongly positive Bull and Breeze Index of 560 (43) and is expected, therefore, to display poor sensitivity upon FAB/MS analysis (42). In a similar manner, GSH conjugates with more than one GSH moiety, such as HPBG, are expected to yield a poor response upon FAB/MS analysis; however, as N - [ (benzyloxy)carbonyl] methyl ester derivatives, these poly-GSH conjugates were detected in samples of biological origin. However, it has been established that the FAB sensitivity of hydrophilic peptides may be enhanced by chemical derivatization to afford a more hydrophobic species (42). As a consequence, therefore, the influence of chemical derivatization upon absolute FAB/MS sensitivity was established for several GSH conjugates either in their native form or as N - [(benzyloxy)carbonyl] methyl ester derivatives. The absolute limit of detection, defined here as the amount of material required to generate an MH+ ion with a signal-to-noise ratio of 3:1, was 1pg (1.6 nmol) for native GSSG (MH+ = 613) and 22.5 ng (24 pmol) for the N-[(benzyloxy)carbonyl]methyl ester derivative (MH+ = 937). These data indicate that a 67-fold increase in absolute sensitivity was observed following the conversion of GSSG to an N-[(benzyloxy)carbonyl] methyl ester derivative prior to FAl3/MS analysis. The general nature of this phenomenon was established by FAB/MS analysis of DHPG and CHPG either in their native forms or in their derivatized forms. In the case of DHPG, a 53-fold gain in sensitivity was observed as the limit of detection was lowered from 3.8 r g (9.9 nmol) for the native conjugate to 100 ng (184 pmol) upon conversion to an N-[(benzyloxy)carbonyl] methyl
1835
ester derivative. In a similar manner, for CHPG, the limit of detection was lowered from 1.5 pg (3.75 m o l ) for the native conjugate to 52.3 ng (93 pmol) upon derivatization, a 40-fold increase in absolute FAB/MS sensitivity. The enhanced sensitivity of derivatized GSH conjugates compared with native GSH conjugates on FAB/MS analysis, their excellent chromatographic properties, and their favorable fragmentation under CID conditions offer considerable benefits for the detection and the characterization of GSH conjugates by tandem mass spectrometry.
ACKNOWLEDGMENT We thank E. A. Lock (IC1 CTL Cheshire, England) for a gift of DCVG used during the course of this investigation. Registry No. DCVG, 2148-32-5; APAP-SG, 64889-81-2; DHPG, 116523-51-4;CHPG, 118744-59-5;HPBG, 126900-04-7; GSSG, 27025-41-8. LITERATURE CITED (1) Chasseeud, L. F. I n Olutethione: Metabolism and Function; Jekoby, W. B., Arias, I. M.. Eds.; Raven: New York. 1976; pp 77-114. (2) Chasseaud, L. F. Drug Metab. Rev. 1973, 2 , 185-220. (3) Jakoby, W. B.; Habig, W. H. I n Enzymatic Basis of Detoxication; Jekoby, W. B., Ed.; Academic Press: New York, 1980; Vol. 11, pp 63-94. Moldeus, P.; Jernstrom, B. In Functions of Glutartrlone, Biochemical, Pbysiokgical, Toxicological, and Clinical Aspects; Larsson. A.. Orrenius, S., Holmgren, A., Mannervik, B., Eds.; Raven: New York, 1983; DO 99-108. Gders. M. W., Ed. Bioactivation of Foreign Compounds; Academlc Press: Orlando. FL. 1985. Koga, N.; Inskekp, P. B.; Harris, T. M.; Guengerich, F. P. Blochemisrty 1988, 2 5 , 2192-2198. Nash, F. A.; King, L. J.; Lock, E. A.; Green, T. Toxicd. Appl. pharmaCOl. 1984, 73, 124-137. Nelson, S. D.; Mitchell, J. R.; Pohl, L. R. In Mass Spechomeby in Drug Metabolism; Frigerio, A., Ghisalberti, E. L., Eds.; Plenum: New York, 1977; pp 237-249. Meerman. J. H. N.; Beland, F. A.; Ketterer, B.; Sral, S. K.; Bruins, A. P.; Mulder, 0. J. Chem. Biol. Interact. 1982, 39, 149-168. Nelson, S.D.; Vaishav, Y.; Kambera, H.; Baillie. T. A. Biomed. Mass. Spectrom. 1981, 8 , 244-251. Tunek, A.; Platt, K. L.; Pryzbylski, M.; Oesch, F. Cbem.-Biol. Interact. 1980, 33, 1-17. Pryzbylski, M.; Cysyk, R. L.; Shoemaker, D.; Ademson, R. H. Biomed. Mass. Spectrom. 1981, 8 , 485-491. Hutson, D. H.; Lakeman, S. K.; Logan, C. J. Xenobiotica 1984, 74, 925-934. Ross, D.; Larsson, R.; Norbeck, K.; Rhyage, R.; Moldeus, P. M i . Pbarmacol. 1985, 2 7 , 277-286. Pallante, S . L.; Lisek, C. A.; Duiik, D. M.; Fenselau, C. Drug Metab. DlSpOS. 1986, 14, 313-318. Foureman, G. L.; Reed, D. J. Biocbemisby 1987, 2 6 , 2028-2033. Haroldson, P. E.; Rellly, M. H.; Hughes, H.; Gaskell, S. J.; Porter, C. J. Blomed. Environ. Mass. Spectrom. 1988, 75, 615-621. Lay, J. 0.;Potter, D. W.; Hinson, J. A. Biomed. Envkon. Mass Specfrom. 1987, 14, 517-521. Pearson, P. G.; Threadgill, M. D.; Howald, W. N.; Balllii, T. A. Biomed. Environ. Mass Spectrom. 1988, 76, 51-56. Threadgill, M. D.; Axworthy, D. 8.; Baillie, T. A,; Farmer, P. B.; Farrow, K. C.; Gescher, A.; Kestell, P.; Pearson, P. G.; Shaw, A. J. J. Pbarmacol. Exp. mer. 1987,242, 312-319. Nocerini, M. R.; Yost, G. S.;Carlson, J. R.; Liberato, D. J.; Breeze, R. G. Drug Metab. Dispos. 1985. 13. 690-694. Sasame, H. A.; Liberto, D. J.; Glllette, J. R. Drug. Metab. Dispos. 1987, 75, 349-355. Parker, C. E.; de Wit, J. S. M.; Smlth, R. W.; Gopinethan, M. 8.; Hernandez, 0.; Tomer, K. 6.; Vestel, C. H.; Sanders, J. M.; Bend, J. R. Biomed. Environ. Mass Spechom. 1988, 15, 623-634. Rashed, M. R.; Pearson, P. G.; Han, D.-H.; Baillie, T. A. Rapid Commun. Mass Spectrom. 1989, 10, 360-363. Bean, M. F.; Pallante-Morell, S. L.; Dulik, D. M.; Fenselau, C. Anal. Cbem. 1990. 62. 121-124. Stock, B. H.i &nd,-J. R ; Eling, T. E. J . Biol. Cbem. 1986, 261, 5959-5964. Stock, B. H.; Schreiber, J.; Guenant, C.; Mason, R. P.; Bend, J. R.; Eling, T. E. J. Biol. Cbem. 1988, 261, 15915-15922. Straub, K. M. I n Mass Spec?rometry in Biomedical Research; Gaskell, S. J., Ed.; J. Wiley & Sons: New York, 1986; pp 115-134. Tomer. K. B.; Guanat, C.; Dino. J. J.; Deterding, L. J. Biomed. Environ. Mass Spectrom. lS88, 76, 473-476. Hoffman, K.J.; Baillie, T. A. Biomed. Environ. Mass Spectrom. 1988, 15, 637-647. Pearson, P. G.; Slater. J. 0.;Rashed. M. R.; Han. D.-H.; Grillo, M. P.; Baiille, T. A. Biochem. Biopbys. Res. Commun. 1989, 766, 245-250. Perchalski, R. J.; Yost, R. A.; Wilder, B. J. Anal Cbem. 1982, 5 4 , 1446-147 1. Lee, M. S.;Yost. R. A. Biomed. Environ. Mass Spectrom. 1988, 15, 193-204. Gaskell, S.J. Biomed. Environ. Mass Spectrom. 1988, 15, 99-104.
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Anal. Chem. 1990, 62, 1836-1840
(35) Mckinney, L. L.; Belster, H. E. J . Am. Chem. Soc. 1959, 88, 909-9 15. (36) Dahlin, D. C.; Nelson, S. D. J . Med. Chem. 1982, 25, 885-886. (37) Pearson, P. G.; Soderlund, E. S.; Dybing, E.; Nelson, S. D. Biochemistw 1990, 20, 4971-4961. (38) Omichinski. J. G.; Nelson, S. D. J . Labelled Compd. Radiopharm. 1988, 25, 263-275. (39) Han, D.-H.; Pearson, P. G.; Dayal, R.; Tsang, L. H.; Baillie, T. A.; Gescher, A. Chem. Res. Toxicol., in press. (40) Jones, T. w.: Gerdes, R. G.; Omstad, K.; Orrenius, s. Chem.-Bio/. Interact. 1885, 56, 251-267. (41) Rashed, M. S.; Nelson, S. D. Chem. Res. Toxicol. 1989, 2 , 41-45. (42) Naylor, S. A.: Findeis, A. F.; Gibson, B. W.; Willims, D. H. J . Am. Chem. SOC. 1988, 108. 6359-6363.
(43) Bull, H. B.; Breeze, K. Arch. Bbchem. Bbphys. 1974, 161, 665-670.
RECEIVED for review February 19, 1990. Accepted May 7,1990. This work presentedin Part at the 37th American Society for Mass Spectrometry Conference on Mass Spectrometry and Allied ~ ~~ i ~~ ~ ~i i was a ~lprovided ~ by a ,grant from the National Institutes of Health (ES02728). The tandem mass spectrometer used in these studies was purchased by grants from the M. J. Murdock Charitable Trust and the National Institutes of Health (RR02262).
High-Accuracy Molecular Mass Determination of Proteins Using Matrix-Assisted Laser Desorption Mass Spectrometry Ronald C. Beavis* and Brian T. Chait The Rockefeller University, New York, New York 10021
A method for obtalnlng protein molecular masses wlth an accuracy of approxknately f0.01% by matrix-assisted laser desorptlon uslng an internal callbrant Is descrlbed. The technique allows accurate mass determinations of proteln sample sires as small as 1 pmol. High concentrations of organlc and inorganic contaminants (e.g. 1 M urea) do not strongly affect either the signal lntenslty or the mass asslgnment. The ability to asslgn an accurate molecular mass to a protein is contingent on the observation of clearly resolved protonated molecule Ions in the mass spectrum.
The matrix-assisted laser desorption technique pioneered by Hillenkamp and Karas ( I , 2) allows the use of a time-offlight mass spectrometer to mass analyze gas-phase protein ions. In the original technique, the protein sample of interest is mixed with a large molar excess of nicotinic acid in solution and then a small amount of this solution is dried onto a sample probe. The probe is then placed into a time-of-flight mass spectrometer, where the surface of the protein/nicotinic acid deposit is irradiated by a short pulse of ultraviolet light from a neodymium-yttrium aluminum garnet laser (Nd-YAG) (frequency quadrupled output, 266 nm). The absorption of the laser light causes the desorption of ions related to both nicotinic acid and the intact protein molecule. The proteinrelated ions produced by this effect are predominantly of charge states z = +1 and +2. It has subsequently been shown that analogous negatively charged ions are produced, with the predominant charge states being z = -1 and -2 (3, 4 ) . The signals produced by the original method using nicotinic acid were characteristically much broader than the instrumental mass resolution of the mass spectrometer used, resulting in a mass resolution ( m / A m ,measured at the full width, half maximum, fwhm) of approximately 50 and a mass accuracy of 0.1% (5). It has been recently shown that one large component of the peak width is caused by a photochemically generated adduction of the nicotinic acid "matrix" molecule to the protein analyte (3). Several new matrix types have been discovered that can be used to desorb protein molecules, which produce much less intense and more easily resolved photochemically generated adducts (6). The best of 0003-2700/90/0362-1836$02.50/0
these matrices are the cinnamic acid derivatives trans-3methoxy-4-hydroxycinnamic acid (ferulic acid) and trans3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid). The extended conjugation of the chromaphore in these materials increases the range of laser wavelengths useful for desorption (compared to nicotinic acid) allowing the use of the frequency tripled output of a Nd-YAG laser (355nm) (7).This wavelength is not absorbed by the analyte molecules of interest, minimizing the possibility of direct absorption of the laser energy by the protein. The use of cinnamic acid derivative matrices, particularly sinapinic acid, for protein analysis has allowed our group to resolve the protonated molecule ion signal from the photochemically generated adduct signal by using a simple, linear time-of-flight mass spectrometer. With the peaks resolved ( m / A m = 300-500, fwhm), the time-of-flight of the centroid of these peaks can be accurately determined. However, to assign the mass that corresponds to this time-of-flight, it is necessary to calibrate the instrument. The relationship between time-of-flight ( t ) and the mass-to-charge ratio ( m / z ) of an ion is given by ( m / z ) 1 / 2= A t
+B
(1)
where A and B are calibration constants that depend on the accelerations given the ion and the ion drift length in the instrument. By knowing the mass-to-charge ratio of any two ions in a spectrum and by measuring their flight times, it is possible to determine A and B uniquely. This technique of calibrating time-of-flight spectra has been used extensively in plasma desorption mass spectrometry (8), secondary ion mass spectrometry (9), and laser desorption mass spectrometry ( l o ) ,where the time-of-flight of two low-mass ions (e.g. [HI+ and [Na]+) are determined accurately and used to extrapolate to higher mass. The use of intense low mass signals to calibrate high mass protein signals w&s originally attempted in our laboratory but was found to be too inaccurate for most applications. The very long flight times of protein ions compared to the low mass calibrants meant that the centroids of the calibrant ions had to be determined very exactly so that the extrapolation to long flight times would be sufficiently accurate. Given the time resolution of the best commercial transient recorders (5 ns), 0 1990 American Chemical Society
