Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
High-performance tandem mass spectrometry in metabolism studies C. Fenselau & P. B. W. Smith To cite this article: C. Fenselau & P. B. W. Smith (1992) High-performance tandem mass spectrometry in metabolism studies, Xenobiotica, 22:9-10, 1207-1219, DOI: 10.3109/00498259209051874 To link to this article: http://dx.doi.org/10.3109/00498259209051874
Published online: 22 Sep 2008.
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Date: 07 May 2016, At: 05:18
XENOBIOTICA,
1992, VOL.22,
NOS
9/10, 1207-1219
High-performance tandem mass spectrometry in metabolism studies
C. FENSELAUt and P. B. W. S M I T H
Downloaded by [Australian National University] at 05:18 07 May 2016
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, M D 21228, USA
Received 20 November 1990; accepted 24 April 1992 1. High-performance tandem mass spectrometry provides unit resolution in both selection of precursor ions and analysis of fragment ions, and extensive and reproducible fragmentation through collisional activation at high energy.
2. Metabolites can be analysed that occur as minor components in h.p.1.c. peaks or other mixtures. Homogeneous isotopic species can be selected for unambiguous analysis of distributions of isotope labels. Fragmentation may be significantly enhanced to provide structural information, Overall, the signal to noise ratio is greatly improved and the spectrum is simplified. 3. These points are illustrated by isotope-labelling studies of the mechanisms of glutathione conjugation of the anti-tumour agent cyclophosphamide, the cytotoxic agent phosphoramide mustard and dimethylbilirubin, an analogue of bilirubin designed to be distinguishable from endogenous bilirubin. Analysis of isomeric mixed disulphides formed between glutathione and a peptide with an internal disulphide bond is discussed. 4. Reaction-induced decomposition is presented as an alternative to collisionally induced decomposition with more efficient energy transfer.
Introduction Tandem mass spectrometry has become one of the most powerful analytical techniques available for analysis of organic and biological molecules. In the usual experiment, ions of a certain mass are selected by transmission through the first mass spectrometer (MS 1) and activated by collision with an inert target gas such as helium. These activated ions undergo decomposition to a set of fragment ions, which are then analysed by the second mass spectrometer (MS 2). Lindholm constructed the first tandem mass spectrometer in 1954, and at least five more were built through the next decade for the study of ion molecule reactions (Lindholm 1972). T h e development of this technique with other analyser configurations was subsequently taken up by analytical laboratories, and its application to analytical problems flowered when instruments with collision cells were offered commercially more than 20 years later (Cooks 1978, McLafferty 1983, Busch et al. 1988). Three configurations of tandem mass spectrometers have been used in drug metabolism and other applications, based on interfacing two quadrupole analysers, two double-focusing analysers, or one of each. T h e second system, whose capabilities will be illustrated here, is also called a four-sector tandem mass spectrometer
t To whom correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd
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C . Fenselau and P. B. W . Smith
because it combines two electrostatic and two magnetic fields. These instruments can analyse ions with unit resolution or better in both the selection of precursor ions and the analysis of fragment ions. Collisional activation is usually carried out at kinetic energies above 2000 eV, which provides highly reproducible fragmentation patterns. These analysers can transmit ions with mass to charge ratios above 8000, although ions this heavy are not readily activated for decomposition. Many investigators have demonstrated the strength of tandem mass spectrometry for analysis of metabolism mixtures, i.e. the use of M S 1 as a separation device (e.g. Richter et al. 1983, Straub 1986, Busch et al. 1988). Molecular ions formed from even a minor component in a mixture can be separated from other components, contaminants and the chemical noise produced by fast atom (FAB) or ion (FIB) bombardment (Fenselau and Cotter 1987). Fragmentation may be significantly enhanced with collisional activation, relative to unimolecular decomposition following desorption. In the latter case, fragment ion peaks are sometimes so weak that they are obscured by the chemical noise. T h e capability of selecting ions with better than unit resolution in MS 1, is particularly powerful for analysis of isotope labels, where incomplete labelling has historically confounded the analysis. These points are illustrated below in studies of metabolism of the antitumour agent cyclophosphamide, the cytotoxic agent phosphoramide mustard and dimethylbilirubin, a bilirubin analogue blocked for normal metabolism.
Mass spectrometry The studies discussed here were carried out on an HXllOHXl10 four-sector tandem mass spectrometer manufactured by Jeol, Tokyo. T h e instrument was fitted with a 10 kV fast atom bombardment source, a collision chamber that can be electrically floated and a DA 5000 computer system for controlling the instrument and acquiring data. Accelerating voltage was set at 10 kV and resolution in both M S 1 and MS 2 was tuned between 500 and 1500. Helium was used in the collision chamber at a pressure that attenuated the ion current by 80%. Collisional activation with helium was carried out at 4000 or 6000 keV. Conjugation of cyclophosphamide and phosphoramide mustard with glutathione As part of on-going studies of the role of glutathione and glutathione transferases in drug resistance acquired by patients receiving therapeutic alkylating agents (Colvin et al. 1988), we have tested a variety of therapeutic alkylating agents as substrates for glutathione transferases. Enzyme catalysis has been observed for the conjugation of chlorambucil (Dulik et al. 1990), and melphalan (Dulik et al. 1986). A new route of conjugation was recognized with melphalan, in which a quaternary ammonium group is displaced by the sulphydryl group (Dulik and Fenselau 1987). More recently we have shown that conjugation of cyclophosphamide is catalysed by both microsomal and cytosolic glutathione transferases, and that its putative active metabolite, phosphoramide mustard, is conjugated by cytosolic enzymes (Yuan et al. 1991). Many nitrogen and sulphur mustards readily form cyclic aziridinium species in aqueous solutions at p H 7 , and we postulated that these intermediates are substrates for the glutathione transferases. T o test this, we used cyclophosphamide and phosphoramide mustard labelled with deuterium in the P-positions of both arms of the mustard moieties. As shown for cyclophosphamide in figure 1, these
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High-performance tandem mass spectrometry in metabolism studies
1 209
isotope labels would be randomized across both the a- and /?-positions if glutathione reacts with the otherwise symmetrical aziridinium ring. Cleavage of the carbon-carbon bond in the mass spectrometer allows the isotope distribution to be analysed. This required cleavage was observed in the mass spectrum of cyclophosphamide monoglutathione obtained by fast atom bombardment. However, the FAB spectrum of the d,-analogue did not lend itself readily to analysis. Figure 2 contains the relevant mass range from the FAB spectra, which can be seen to be complicated by chlorine isotopes and other signals. Figure 3 shows this same mass range from a spectrum obtained by collisional activation of molecular ions of cyclophosphamide monoglutathione that contain only C1-3 5 and four deuterium atoms. It can be clearly seen that selection of a monoisotopic parent ion clarifies the isotope pattern and that the fragment ion retains all four labels, consistent with direct displacement of chlorine anions as the mechanism of conjugation. Phosphoramide mustard is known to form a cyclic aziridinium intermediate more readily than cyclophosphamide at p H 7.0. Again, deuterium isotope labels were used to test the mechanism of conjugation with glutathione. T h e strategy again requires C-C cleavage on the mass spectrometry as indicated in figure 4. However, fragment ions produced by this cleavage were not visible above the noise in the FAB anion and cation spectra of the diglutathione formed from phosphoramide mustard. Nor did collisional activation of molecular ion species produce these fragment ions in a tandem mass spectrum experiment. However, collisional activation of a fragment ion formed in the FAB source and selected by MS 1 provided ions comprising both pieces formed by cleavage of the carbon-carbon bond. T h e genealogy of the ions analysed is shown in figure 5. Figure 6 presents the regions of interest in spectra obtained by collisional activation of the mass 684 and 688 fragment ions formed from labelled and unlabelled conjugates. T h e fragment ions of interest stand out clearly against background noise in the spectrum. In contrast to the situation with cyclophosphamide, here the isotope labelling evidence supports conjugation of a symmetrical intermediate such as the
Figure 1.
The path of deuterium labels in two mechanisms of conjugation between glutathione and d,-cyclophosphamide.
C . Fenselau and P . B . W . Smith
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Figure 2.
Partial fast atom bombardment mass spectra of the monoglutathione conjugates of cyclophosphamide and d,-cyclophosphamide.
aziridinium zwitterion. A secondary isotope effect could be detected in both of the ions analysed (Yuan et al. 1991).
Metabolism of dimethylbilirubin T h e major excretory pathway for bilirubin involves conjugation with glucuronic acid. When this transformation is impaired, e.g. genetically as in CriglerNajjar syndrome or the Gunn rat, or by disease, the resulting hyperbilirubinaemia
100
.-
80
.-
Q,
A.
0
c
0 0 C
60.-
1
n
a al
40.-
> .-c
-0 Q,
E
20.-
I
0 210
2 20
210
220
m/z Figure 3. Partial collisional activation mass spectra of the monoglutathione conjugates of (A) cyclophosphamide and (B) d,-cyclophosphamide obtained on a four-sector tandem mass spectrometer. Adapted from Yuan et al. 1991.
High-performance tandem mass spectrometry in metabolism studies
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1
121 1
7
R
0-
L
Figure 4. The path of deuterium labels in two mechanisms of conjugation between glutathione and d,-phosphoramide mustard.
is life-threatening. Alternative pathways of metabolism and excretion of bilirubin and its analogues have been detected in the Gunn rat, most dramatically following induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Ode11 et al. 1992). We undertook to identify the alternative metabolites and the responsible enzyme systems, using dimethylbilirubin as an analogue of bilirubin (figure 7) whose metabolites can be distinguished from endogenous bile pigments. Metabolites were purified from bile from cannulated Gunn and Wistar rats. Comparison of the high-pressure liquid chromatograms of biliary metabolites of dimethylbilirubin formed by Gunn rats indicated a series of metabolites that were not conjugated to glucuronic acid. Molecular weights obtained by plasma
lH+
1
H+
OH
m / r 763
m/z 684
m/z 321
m/z 363
G S = glutathionyl Figure 5.
Formation of the product ions analysed from the diglutathione conjugate of phosphoramide mustard and its d,-analogue.
C . Fenselau and P. B. W . Smith
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--
I
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36
365
5
367
--
310
320
330
340
350
360
370
m/z Figure 6. Partial collisional activation spectrum of the diglutathione conjugates of phosphoramide mustard and d,-phosphoramide mustard obtained on a four-sector tandem mass spectrometer. Adapted from Yuan et al. 1991.
Figure 7.
Bilirubin
R=R'=H
Dimethylbilirubin
R=R=CH3
Bilirubin C-12-glucuronide
R=H,R=C,5H9O,j
Bilirubin derivatives and products of cleavages on either site of the central methylene group.
High-performance tandem mass spectrometry in metabolism studies
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FAB MSMS scan (936) of bilirubin diglucuronide
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I0
z Figure 8. Collisional activation spectrum of bilirubin diglucuronide obtained on a four-sector tandem mass spectrometer.
desorption mass spectrometry (Fenselau et al. 1989) indicated the presence of at least three isomers of mass 935.5 u, and the loss of 306 u as a neutral fragment ion suggested conjugation with glutathione. T h e presence of glutathione was subsequently supported by treatment with y-glutamyltranspeptidase, amino acid analysis and n.m.r. N.m.r. studies indicated that the C-18 exocyclic vinyl group was the site of conjugation. T h e molecular weight also required addition of another 17 u to the metabolites, whose formation is induced by T C D D . T h e spectra of bilirubin glucuronides and of the isomeric metabolites of dimethylbilirubin were obtained with collisional activation on the highperformance tandem mass spectrometer as shown in figures 8 and 9. T h e simplicity of the fragmentation pattern is startling, and it is highly diagnostic of the structural changes due to metabolism. T h e tetrapyrrole is cleaved on either side of the central methylene group, as shown in figure 7, and the resulting ions allow symmetrical assignment of the two glucuronide moieties (figure 8), and unsymmetrical assignment (both on the same half of the molecule) of glutathione and oxygen in the metabolite of dimethylbilirubin (figure 9). Neutral losses characterize the nature of conjugates directly. T h e loss of dehydroglucuronic acid, 176 u, is characteristic for underivatized glucuronides as a class (Fenselau et al. 1983). T h e loss of 306u from the glutathione conjugate, cleavage of the C-S bond accompanied by proton transfer, is one of a set of class characteristic cleavages that occur in glutathione conjugates under high-energy collisional activation. T h e additional hydroxyl group is also detected in the elimination of water, 18u. An important feature of the spectrum in figure 9 is evidence to support assignment of glutathionyl and hydroxyl groups to the same halves of the tetrapyrrole. Figure 10 shows the fast atom bombardment spectrum of one peak collected from the high-pressure liquid chromatograph. T h e protonated molecular ion at mass 936 is visible, accompanied by the Na (m/z 958) and K (unlabelled, m/z 974) satellites. A small peak is also visible at mlz952. This minor bile metabolite was selected by MS 1, activated and analysed by MS 2 in a tandem mass spectrometry experiment (figure 11). Although the signal to noise ratio is weak, the spectrum indicates that this bilirubin derivative carries a second oxygen atom, attached to the other half of the tetrapyrrole. T C D D induction and an understanding of the substrate requirements for glutathione transferases led to the hypothesis that epoxidation of the exocyclic
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vinyl group is catalysed by a monooxygenase(s) in the P-450 family, followed by conjugation with glutathione. (This sequence provides opportunity for formation of stereo- and regio-isomers.) T o test the involvement of a monooxygenase, the metabolism of dimethylbilirubin was carried out in nitro, using Gunn rat hepatic microsomes after induction with T C D D (Shore et al. 1989), under an atmosphere enriched in T h e tandem mass spectrum in figure 12 may be compared with that in figure 9 to confirm that one atom from molecular oxygen is incorporated into the same half of the tetrapyrrole as glutathione (Shore et al. 1992).
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Mixed disulphides involving glutathione Tandem mass spectrometry, particularly employing a four-sector instrument, is best known for sequencing peptides. T h e fragment ions formed by high-energy collisional activation have been found to be reproducible from one laboratory to another, and to comprise, for the most part, ions formed by one of less than a dozen cleavage patterns centred around each amide bond in the polymer backbone (Biemann 1988, Johnson et al. 1988). T h e generally accepted nomenclature for six of these is illustrated in figure 13. This technique can readily distinguish the
Q)
0
E
m
za
2
i'
C
(1-a)
!O) .3
15
1;.
e MIZ Figure 9. Collisional activation spectrum of the glutathione conjugated metabolite of bilirubin dimethyl ester (M H = 936.5) obtained on a four-sector tandem mass spectrometer.
+
Adapted from Ode11 et al. 1992.
High-performance tandem mass spectrometry in metabolism studies
1215
sr
FAB MS (quadratic scan) of dimethylbilirubin pigmenl # I
952.5
*8.0'
L , I ..h '00
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a
400
500
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I
. 800
'
900
I .
1000
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M/Z
Figure 10. Fast atom bombardment mass spectrum of an h.p.1.c. peak collected in the purification of metabolites of dimethyl bilirubin.
isomeric mixed disulphides formed by chemical reaction between glutathione and vasopressin and purified by h.p.1.c. These spectra are shown in figure 14, with sequence and other ions annotated. T h e structure assigned is also indicated on each spectrum. Sequence ions are identified at intervals across the spectrum in figure 14a, reflecting mass increments associated with residues 1 to 8. By contrast, a gap of about 300amu exists in the middle of the spectrum in figure 14b, reflecting the attachment of glutathione to cysteine-6. Sequence ions are observed at the expected mass increments for residues 1 4 and 7. In both spectra residues 8 and 9 are characterized together in the x2 ion. Arginine near the carboxy terminus localizes the ionizing proton, and thus most of the sequence ions detected are those that contain the carboxy terminus-v, w , x, y, z. Cleavage in the protonated molecular ions also occurs in three places across the disulphide linkage, contributing abundant ions in the mass range 10001125 in both spectra (figure 15). Cleavage within the tripeptide glutathione may contribute ions of mass 1261 by loss of the glutamyl group. Some single-residue immonium ions (Biemann 1988) are labelled below mass 150 in both spectra.
2 1 a
t
-1 '
FAB MSMS scan (952) of dimethylbilirubin pigment # 1
4-
" G
3-
d
a
2-
30
M/2
Figure 11. Collisional activation spectrum of a minor metabolite, M
+ H = 952 in figure 10.
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C . Fenselau and P . B. W . Smith 2o
P
938.5
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8 5 15
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1
C
51
a 10
-8aa> w
(A
z 5 U
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0
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3 800 800
Figure 12. Collisional activation spectrum of a metabolite of bilirubin dimethyl ester formed in vitro in an ''0, atmosphere. Adapted from Shore et al. 1992.
Reaction-induced decomposition Despite the unique kind of analytical capability illustrated in figure 14, highenergy collisional activation does have some limits. These include very inefficient conversion of kinetic energy into vibrational energy and fragmentation, complex fragmentation, and the inability to fragment heavy ions (Orlando et al. 1990 d). An alternative approach under development in our laboratory has been shown to provide highly efficient energy transfer and to offer potential improvements for the other two problems as well. I n this technique a gas is used in the collision cell, which undergoes a slightly endothermic proton transfer reaction with the protonated molecular ion of the sample (Orlando et al. 1989). Projectile ions are slowed to energies in the low eV range, just sufficient to overcome the endothermicity of the reaction. In this circumstance sticky collisions take place, in which complexes are formed between the projectile ion and the target gas molecule. Within these complexes, excess energy is vibrationally randomized, including small amounts of kinetic energy beyond the threshold value. Reactive (endothermic) collisions taking place at 10 eV, for example, lead to spectra that are similar to those recorded for 2000 eV collisions with helium, and very different from those recorded for 30eV collisions with ammonia or helium (Orlando et al. 1990a). We have also found that different bonds are fragmented in different ranges of kinetic energy
0
HZN-
Figure 13.
11 -C-
-OH
Six scissions of peptide bonds potentially generated by collisional activation.
Bieman 1988.
High-performance tandem mass spectrometry in metabolism studies
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YGLU-CYS-GLY
I
H-CYS-TYR-PHE-GLN-ASN-CYS-PRO-ARG-GLY-NHz
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M/Z - ... . . . . _. . . . .. . . . . .. . . Figure 14. Collisional activation spectra ot isomeric mixed disulphides tormed between glutathione and vasopressin.
(Orlando et al. 1990b). For example, in figure 16 the sticky complex formed between ammonia and N,N,N-triacetyl chitotriose is formed when the protonated trisaccharide is decelerated to provide collisions between 3 and 7eV, while cleavage of bonds adjacent to the amide group (which presumably is the site of the endothermic reaction with ammonia) occurs at energies between 10 and 14eV. At slightly higher kinetic energies (>15eV) ions are formed by cleavage in the carbohydrate linkages (Orlando et al. 1990 c). T h e potential application to glycopeptides and other glycosides is obvious.
Acknowledgements Research in the Structural Biochemistry Center at UMBC was supported by the National Science Foundation and the National Institutes of Health. H
I0 R-
Figure 15.
s
1
CHJS SIGH., - R '
Class characteristic cleavage in disulphide linkages.
C. Fenselau and P . B . W. Smith
1218 200
,
u
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-
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--n 0 C
1 204 (M+NH4lxlO NH4+x100
586(x50) 60(x50) 407 (x 1/21
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NH
I
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40
I
c:o
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CHI
I
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Figure 16. Abundances of fragment ions formed by collisions between ammonia and N,N,N-triacetyl chitotriose at energies low enough to permit formation of complexes for endothermic reactions. Adapted from Orlando et al. 1990 c.
References BIEMANN, K., 1988,Contributions of mass spectrometry to peptide and protein structure. Biomedical and Environmental Mass Spectrometry, 16, 99-1 11. BUSCH,K.L., GLISH,G. L., and MCLUCKEY, S. A., 1988,Mass SpectrometrylMass Spectrometry (New York: VCH). M., Russo, J. E., HILTON,J., DULIK,D. M., and FENSELAU, C., 1988,Enzymatic mechanisms COLVIN, of resistance to alkylating agents in tumor cells and normal tissues. Adwances in Enzyme Regulation, 21, 21 1-221. COOKS,R. G. (ed.), 1978,Collision Spectroscopy (New York: Plenum Press). C., 1987,Conversion of melphalan to 4-(glutathionyl)-phenylalanine: a DULIK,D. M., and FENSELAU, novel mechanism for conjugation by glutathione-S-transferases. Drug Metabolism and Disposition, 15, 195-199. C., 1990, Characterization of glutathione conjugates of. DULIK,D., COLVIN,M., and FENSELAU, chlorambucil by fast atom bombardment and thermospray liquid chromatography/mass spectrometry. Biomedical and Environmental Mass Spectrometry, 19, 248-252. DULIK,D. M., FENSELAU, C., and HILTON,J., 1986. Characterization of melphalan-GSH adducts whose formation is catalyzed by glutathione transferase. Biochemical Pharmacology, 35,
3405-3410. FENSELAU, C . , and COTTER,R. J., 1987, Chemical aspects of fast atom mass spectrometry. Chemical Rwiews, 81, 501-512. FENSELAU, C., WANG,R., ODELL,G. B., and MOGILEVSKY, W., 1989,Plasma desorption characterization of glucuronide and glutathione conjugates derived from bilirubin. International Journal of Mass Spectrometry. Ion Processes, 92, 289-295. FENSELAU, C., YELLE, L., STOGNIEW, M., LIBERATO, D., LEHMAN, D., FENG,P., and COLVIN, M., 1983, Analysis of glucuronides by fast atom bombardment. International Journal of Mass Spectrometry und Ion Physics, 46, 41 1414. JOHNSON, R. S.,MARTIN, S. A., and BIEMANN, K., 1988,Collision-induced fragmentation of (M+H)* ions of peptides. Side chain specific sequence ions. fnternational Journal of Mass Spectrometry. Ion Processes, 86, 137-154. LINDHOLM, E.,1972,Mass spectra and appearance potentials studied by the use of charge exchange in a tandem mass spectrometer, in Ion-Molecule Reactions, vol. 2, edited by J. L. Franklin (New York: Plenum Press), pp. 457484. MCLAFFERTY, F. W. (ed.), 1983, Tandem-mass Spectrometry (New York: Wiley Interscience). ODELL,G . B., MOGILEVSKY, W. S., SMITH,P. B. W., and FENSELAU, C., 1991,The identification of glutathione conjugates of the dimethyl ester of bilirubin in the bile of Gunn rats. Molecular Pharmacology, 40, 597-605.
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ORLANDO, R., FENSELAU, C., and COTTER,R. J., 1989, Endothermic ion molecule reactions: 11. Reactions between peptides with and without basic residues and ammonia. Organic Mass Spectrometry, 24, 1033-1042. ORLANDO, R., FENSELAU, C., and COTTER, R. J., 1990a, Endothermic ion-molecule reactions: 111. High energy collisional activation at low kinetic energies. Rapid Communications in Mass Spectrometry, 4, 259-262. ORLANDO, R., FENSELAU, C., and COTTER,R. J., 1990 b, Endothermic ion-molecule reactions. V. Remote-site fragmentation at very low kinetic energies. Organic Mass Spectrometry, 20, 485-489. ORLANDO, R., FENSELAU, C., and COTTER, R. J., 1990c, Endothermic ion-molecule reactions: IV. Sitedirected fragmentation in N-acetylated oligosaccharides at low beam energies. Analytical Chemistry, 62, 2388-2390. ORLANDO, R., MURPHY, C., FENSELAU, C., HANSEN, G., and COTTER, R. J., 1990d, Endothermic ionmolecule reactions: strategies for Tandem Mass Spectrometric structural analyses of large biomolecules. Analytical Chemistry, 62, 125-129. RICHTER, W. J., BLUM,W., SCHLUNEGGER, U. P., and SENN,M., 1983, Tandem mass spectrometry of pharmaceuticals, in Tandem Mass Spectrometry, edited by F. W. McLafferty (New York: Wiley Interscience), pp. 417-434. SHORE, L. S., MOGILEVSKY, W. S., and ODELL,G . B., 1989, T h e in oitro formation of glutathione conjugates of bilirubin dimethylester by hepatic microsomes of jaundiced Gunn rats. Hepatology, 10, 615(A). SHORE, L., MOGILEVSKY, W., SMITH, P., FENSELAU, C., and ODELL,G . , 1991, The in oitro formation of glutathione conjugates of the dimethylester of bilirubin. Biochemical Pharmacology, 42, 1969-1976. STRAUB, K. M., 1986, Metabolic mapping of drugs: rapid screening for polar metabolites using FAB MS/MS, in Mass Spectrometry in Biomedical Research, edited by S. Gaskill (Chichester: John Wiley and Sons). YUAN,2. M., SMITH, P. B., BRUNDRETT, R . B., COLVIN, M., and FENSELAU, C., 1991, Glutathione conjugation with phosphoramide mustard and cyclophosphamide: a mechanistic studying using tandem mass spectrometry. Drug Metabolism and Disposition, 19, 625-629.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
High-performance tandem mass spectrometry in metabolism studies C. Fenselau & P. B. W. Smith To cite this article: C. Fenselau & P. B. W. Smith (1992) High-performance tandem mass spectrometry in metabolism studies, Xenobiotica, 22:9-10, 1207-1219, DOI: 10.3109/00498259209051874 To link to this article: http://dx.doi.org/10.3109/00498259209051874
Published online: 22 Sep 2008.
Submit your article to this journal
Article views: 14
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [Australian National University]
Date: 07 May 2016, At: 05:18
XENOBIOTICA,
1992, VOL.22,
NOS
9/10, 1207-1219
High-performance tandem mass spectrometry in metabolism studies
C. FENSELAUt and P. B. W. S M I T H
Downloaded by [Australian National University] at 05:18 07 May 2016
Department of Chemistry and Biochemistry, University of Maryland Baltimore County, Baltimore, M D 21228, USA
Received 20 November 1990; accepted 24 April 1992 1. High-performance tandem mass spectrometry provides unit resolution in both selection of precursor ions and analysis of fragment ions, and extensive and reproducible fragmentation through collisional activation at high energy.
2. Metabolites can be analysed that occur as minor components in h.p.1.c. peaks or other mixtures. Homogeneous isotopic species can be selected for unambiguous analysis of distributions of isotope labels. Fragmentation may be significantly enhanced to provide structural information, Overall, the signal to noise ratio is greatly improved and the spectrum is simplified. 3. These points are illustrated by isotope-labelling studies of the mechanisms of glutathione conjugation of the anti-tumour agent cyclophosphamide, the cytotoxic agent phosphoramide mustard and dimethylbilirubin, an analogue of bilirubin designed to be distinguishable from endogenous bilirubin. Analysis of isomeric mixed disulphides formed between glutathione and a peptide with an internal disulphide bond is discussed. 4. Reaction-induced decomposition is presented as an alternative to collisionally induced decomposition with more efficient energy transfer.
Introduction Tandem mass spectrometry has become one of the most powerful analytical techniques available for analysis of organic and biological molecules. In the usual experiment, ions of a certain mass are selected by transmission through the first mass spectrometer (MS 1) and activated by collision with an inert target gas such as helium. These activated ions undergo decomposition to a set of fragment ions, which are then analysed by the second mass spectrometer (MS 2). Lindholm constructed the first tandem mass spectrometer in 1954, and at least five more were built through the next decade for the study of ion molecule reactions (Lindholm 1972). T h e development of this technique with other analyser configurations was subsequently taken up by analytical laboratories, and its application to analytical problems flowered when instruments with collision cells were offered commercially more than 20 years later (Cooks 1978, McLafferty 1983, Busch et al. 1988). Three configurations of tandem mass spectrometers have been used in drug metabolism and other applications, based on interfacing two quadrupole analysers, two double-focusing analysers, or one of each. T h e second system, whose capabilities will be illustrated here, is also called a four-sector tandem mass spectrometer
t To whom correspondence should be addressed. 0049-8254/92 $3.00 0 1992 Taylor & Francis Ltd
Downloaded by [Australian National University] at 05:18 07 May 2016
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C . Fenselau and P. B. W . Smith
because it combines two electrostatic and two magnetic fields. These instruments can analyse ions with unit resolution or better in both the selection of precursor ions and the analysis of fragment ions. Collisional activation is usually carried out at kinetic energies above 2000 eV, which provides highly reproducible fragmentation patterns. These analysers can transmit ions with mass to charge ratios above 8000, although ions this heavy are not readily activated for decomposition. Many investigators have demonstrated the strength of tandem mass spectrometry for analysis of metabolism mixtures, i.e. the use of M S 1 as a separation device (e.g. Richter et al. 1983, Straub 1986, Busch et al. 1988). Molecular ions formed from even a minor component in a mixture can be separated from other components, contaminants and the chemical noise produced by fast atom (FAB) or ion (FIB) bombardment (Fenselau and Cotter 1987). Fragmentation may be significantly enhanced with collisional activation, relative to unimolecular decomposition following desorption. In the latter case, fragment ion peaks are sometimes so weak that they are obscured by the chemical noise. T h e capability of selecting ions with better than unit resolution in MS 1, is particularly powerful for analysis of isotope labels, where incomplete labelling has historically confounded the analysis. These points are illustrated below in studies of metabolism of the antitumour agent cyclophosphamide, the cytotoxic agent phosphoramide mustard and dimethylbilirubin, a bilirubin analogue blocked for normal metabolism.
Mass spectrometry The studies discussed here were carried out on an HXllOHXl10 four-sector tandem mass spectrometer manufactured by Jeol, Tokyo. T h e instrument was fitted with a 10 kV fast atom bombardment source, a collision chamber that can be electrically floated and a DA 5000 computer system for controlling the instrument and acquiring data. Accelerating voltage was set at 10 kV and resolution in both M S 1 and MS 2 was tuned between 500 and 1500. Helium was used in the collision chamber at a pressure that attenuated the ion current by 80%. Collisional activation with helium was carried out at 4000 or 6000 keV. Conjugation of cyclophosphamide and phosphoramide mustard with glutathione As part of on-going studies of the role of glutathione and glutathione transferases in drug resistance acquired by patients receiving therapeutic alkylating agents (Colvin et al. 1988), we have tested a variety of therapeutic alkylating agents as substrates for glutathione transferases. Enzyme catalysis has been observed for the conjugation of chlorambucil (Dulik et al. 1990), and melphalan (Dulik et al. 1986). A new route of conjugation was recognized with melphalan, in which a quaternary ammonium group is displaced by the sulphydryl group (Dulik and Fenselau 1987). More recently we have shown that conjugation of cyclophosphamide is catalysed by both microsomal and cytosolic glutathione transferases, and that its putative active metabolite, phosphoramide mustard, is conjugated by cytosolic enzymes (Yuan et al. 1991). Many nitrogen and sulphur mustards readily form cyclic aziridinium species in aqueous solutions at p H 7 , and we postulated that these intermediates are substrates for the glutathione transferases. T o test this, we used cyclophosphamide and phosphoramide mustard labelled with deuterium in the P-positions of both arms of the mustard moieties. As shown for cyclophosphamide in figure 1, these
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1 209
isotope labels would be randomized across both the a- and /?-positions if glutathione reacts with the otherwise symmetrical aziridinium ring. Cleavage of the carbon-carbon bond in the mass spectrometer allows the isotope distribution to be analysed. This required cleavage was observed in the mass spectrum of cyclophosphamide monoglutathione obtained by fast atom bombardment. However, the FAB spectrum of the d,-analogue did not lend itself readily to analysis. Figure 2 contains the relevant mass range from the FAB spectra, which can be seen to be complicated by chlorine isotopes and other signals. Figure 3 shows this same mass range from a spectrum obtained by collisional activation of molecular ions of cyclophosphamide monoglutathione that contain only C1-3 5 and four deuterium atoms. It can be clearly seen that selection of a monoisotopic parent ion clarifies the isotope pattern and that the fragment ion retains all four labels, consistent with direct displacement of chlorine anions as the mechanism of conjugation. Phosphoramide mustard is known to form a cyclic aziridinium intermediate more readily than cyclophosphamide at p H 7.0. Again, deuterium isotope labels were used to test the mechanism of conjugation with glutathione. T h e strategy again requires C-C cleavage on the mass spectrometry as indicated in figure 4. However, fragment ions produced by this cleavage were not visible above the noise in the FAB anion and cation spectra of the diglutathione formed from phosphoramide mustard. Nor did collisional activation of molecular ion species produce these fragment ions in a tandem mass spectrum experiment. However, collisional activation of a fragment ion formed in the FAB source and selected by MS 1 provided ions comprising both pieces formed by cleavage of the carbon-carbon bond. T h e genealogy of the ions analysed is shown in figure 5. Figure 6 presents the regions of interest in spectra obtained by collisional activation of the mass 684 and 688 fragment ions formed from labelled and unlabelled conjugates. T h e fragment ions of interest stand out clearly against background noise in the spectrum. In contrast to the situation with cyclophosphamide, here the isotope labelling evidence supports conjugation of a symmetrical intermediate such as the
Figure 1.
The path of deuterium labels in two mechanisms of conjugation between glutathione and d,-cyclophosphamide.
C . Fenselau and P . B . W . Smith
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Figure 2.
Partial fast atom bombardment mass spectra of the monoglutathione conjugates of cyclophosphamide and d,-cyclophosphamide.
aziridinium zwitterion. A secondary isotope effect could be detected in both of the ions analysed (Yuan et al. 1991).
Metabolism of dimethylbilirubin T h e major excretory pathway for bilirubin involves conjugation with glucuronic acid. When this transformation is impaired, e.g. genetically as in CriglerNajjar syndrome or the Gunn rat, or by disease, the resulting hyperbilirubinaemia
100
.-
80
.-
Q,
A.
0
c
0 0 C
60.-
1
n
a al
40.-
> .-c
-0 Q,
E
20.-
I
0 210
2 20
210
220
m/z Figure 3. Partial collisional activation mass spectra of the monoglutathione conjugates of (A) cyclophosphamide and (B) d,-cyclophosphamide obtained on a four-sector tandem mass spectrometer. Adapted from Yuan et al. 1991.
High-performance tandem mass spectrometry in metabolism studies
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1
121 1
7
R
0-
L
Figure 4. The path of deuterium labels in two mechanisms of conjugation between glutathione and d,-phosphoramide mustard.
is life-threatening. Alternative pathways of metabolism and excretion of bilirubin and its analogues have been detected in the Gunn rat, most dramatically following induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Ode11 et al. 1992). We undertook to identify the alternative metabolites and the responsible enzyme systems, using dimethylbilirubin as an analogue of bilirubin (figure 7) whose metabolites can be distinguished from endogenous bile pigments. Metabolites were purified from bile from cannulated Gunn and Wistar rats. Comparison of the high-pressure liquid chromatograms of biliary metabolites of dimethylbilirubin formed by Gunn rats indicated a series of metabolites that were not conjugated to glucuronic acid. Molecular weights obtained by plasma
lH+
1
H+
OH
m / r 763
m/z 684
m/z 321
m/z 363
G S = glutathionyl Figure 5.
Formation of the product ions analysed from the diglutathione conjugate of phosphoramide mustard and its d,-analogue.
C . Fenselau and P. B. W . Smith
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--
I
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36
365
5
367
--
310
320
330
340
350
360
370
m/z Figure 6. Partial collisional activation spectrum of the diglutathione conjugates of phosphoramide mustard and d,-phosphoramide mustard obtained on a four-sector tandem mass spectrometer. Adapted from Yuan et al. 1991.
Figure 7.
Bilirubin
R=R'=H
Dimethylbilirubin
R=R=CH3
Bilirubin C-12-glucuronide
R=H,R=C,5H9O,j
Bilirubin derivatives and products of cleavages on either site of the central methylene group.
High-performance tandem mass spectrometry in metabolism studies
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FAB MSMS scan (936) of bilirubin diglucuronide
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I0
z Figure 8. Collisional activation spectrum of bilirubin diglucuronide obtained on a four-sector tandem mass spectrometer.
desorption mass spectrometry (Fenselau et al. 1989) indicated the presence of at least three isomers of mass 935.5 u, and the loss of 306 u as a neutral fragment ion suggested conjugation with glutathione. T h e presence of glutathione was subsequently supported by treatment with y-glutamyltranspeptidase, amino acid analysis and n.m.r. N.m.r. studies indicated that the C-18 exocyclic vinyl group was the site of conjugation. T h e molecular weight also required addition of another 17 u to the metabolites, whose formation is induced by T C D D . T h e spectra of bilirubin glucuronides and of the isomeric metabolites of dimethylbilirubin were obtained with collisional activation on the highperformance tandem mass spectrometer as shown in figures 8 and 9. T h e simplicity of the fragmentation pattern is startling, and it is highly diagnostic of the structural changes due to metabolism. T h e tetrapyrrole is cleaved on either side of the central methylene group, as shown in figure 7, and the resulting ions allow symmetrical assignment of the two glucuronide moieties (figure 8), and unsymmetrical assignment (both on the same half of the molecule) of glutathione and oxygen in the metabolite of dimethylbilirubin (figure 9). Neutral losses characterize the nature of conjugates directly. T h e loss of dehydroglucuronic acid, 176 u, is characteristic for underivatized glucuronides as a class (Fenselau et al. 1983). T h e loss of 306u from the glutathione conjugate, cleavage of the C-S bond accompanied by proton transfer, is one of a set of class characteristic cleavages that occur in glutathione conjugates under high-energy collisional activation. T h e additional hydroxyl group is also detected in the elimination of water, 18u. An important feature of the spectrum in figure 9 is evidence to support assignment of glutathionyl and hydroxyl groups to the same halves of the tetrapyrrole. Figure 10 shows the fast atom bombardment spectrum of one peak collected from the high-pressure liquid chromatograph. T h e protonated molecular ion at mass 936 is visible, accompanied by the Na (m/z 958) and K (unlabelled, m/z 974) satellites. A small peak is also visible at mlz952. This minor bile metabolite was selected by MS 1, activated and analysed by MS 2 in a tandem mass spectrometry experiment (figure 11). Although the signal to noise ratio is weak, the spectrum indicates that this bilirubin derivative carries a second oxygen atom, attached to the other half of the tetrapyrrole. T C D D induction and an understanding of the substrate requirements for glutathione transferases led to the hypothesis that epoxidation of the exocyclic
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vinyl group is catalysed by a monooxygenase(s) in the P-450 family, followed by conjugation with glutathione. (This sequence provides opportunity for formation of stereo- and regio-isomers.) T o test the involvement of a monooxygenase, the metabolism of dimethylbilirubin was carried out in nitro, using Gunn rat hepatic microsomes after induction with T C D D (Shore et al. 1989), under an atmosphere enriched in T h e tandem mass spectrum in figure 12 may be compared with that in figure 9 to confirm that one atom from molecular oxygen is incorporated into the same half of the tetrapyrrole as glutathione (Shore et al. 1992).
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Mixed disulphides involving glutathione Tandem mass spectrometry, particularly employing a four-sector instrument, is best known for sequencing peptides. T h e fragment ions formed by high-energy collisional activation have been found to be reproducible from one laboratory to another, and to comprise, for the most part, ions formed by one of less than a dozen cleavage patterns centred around each amide bond in the polymer backbone (Biemann 1988, Johnson et al. 1988). T h e generally accepted nomenclature for six of these is illustrated in figure 13. This technique can readily distinguish the
Q)
0
E
m
za
2
i'
C
(1-a)
!O) .3
15
1;.
e MIZ Figure 9. Collisional activation spectrum of the glutathione conjugated metabolite of bilirubin dimethyl ester (M H = 936.5) obtained on a four-sector tandem mass spectrometer.
+
Adapted from Ode11 et al. 1992.
High-performance tandem mass spectrometry in metabolism studies
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sr
FAB MS (quadratic scan) of dimethylbilirubin pigmenl # I
952.5
*8.0'
L , I ..h '00
200
a
400
500
600
700
I
. 800
'
900
I .
1000
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M/Z
Figure 10. Fast atom bombardment mass spectrum of an h.p.1.c. peak collected in the purification of metabolites of dimethyl bilirubin.
isomeric mixed disulphides formed by chemical reaction between glutathione and vasopressin and purified by h.p.1.c. These spectra are shown in figure 14, with sequence and other ions annotated. T h e structure assigned is also indicated on each spectrum. Sequence ions are identified at intervals across the spectrum in figure 14a, reflecting mass increments associated with residues 1 to 8. By contrast, a gap of about 300amu exists in the middle of the spectrum in figure 14b, reflecting the attachment of glutathione to cysteine-6. Sequence ions are observed at the expected mass increments for residues 1 4 and 7. In both spectra residues 8 and 9 are characterized together in the x2 ion. Arginine near the carboxy terminus localizes the ionizing proton, and thus most of the sequence ions detected are those that contain the carboxy terminus-v, w , x, y, z. Cleavage in the protonated molecular ions also occurs in three places across the disulphide linkage, contributing abundant ions in the mass range 10001125 in both spectra (figure 15). Cleavage within the tripeptide glutathione may contribute ions of mass 1261 by loss of the glutamyl group. Some single-residue immonium ions (Biemann 1988) are labelled below mass 150 in both spectra.
2 1 a
t
-1 '
FAB MSMS scan (952) of dimethylbilirubin pigment # 1
4-
" G
3-
d
a
2-
30
M/2
Figure 11. Collisional activation spectrum of a minor metabolite, M
+ H = 952 in figure 10.
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C . Fenselau and P . B. W . Smith 2o
P
938.5
Y'
8 5 15
2a
1
C
51
a 10
-8aa> w
(A
z 5 U
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0
200
0
400
3 800 800
Figure 12. Collisional activation spectrum of a metabolite of bilirubin dimethyl ester formed in vitro in an ''0, atmosphere. Adapted from Shore et al. 1992.
Reaction-induced decomposition Despite the unique kind of analytical capability illustrated in figure 14, highenergy collisional activation does have some limits. These include very inefficient conversion of kinetic energy into vibrational energy and fragmentation, complex fragmentation, and the inability to fragment heavy ions (Orlando et al. 1990 d). An alternative approach under development in our laboratory has been shown to provide highly efficient energy transfer and to offer potential improvements for the other two problems as well. I n this technique a gas is used in the collision cell, which undergoes a slightly endothermic proton transfer reaction with the protonated molecular ion of the sample (Orlando et al. 1989). Projectile ions are slowed to energies in the low eV range, just sufficient to overcome the endothermicity of the reaction. In this circumstance sticky collisions take place, in which complexes are formed between the projectile ion and the target gas molecule. Within these complexes, excess energy is vibrationally randomized, including small amounts of kinetic energy beyond the threshold value. Reactive (endothermic) collisions taking place at 10 eV, for example, lead to spectra that are similar to those recorded for 2000 eV collisions with helium, and very different from those recorded for 30eV collisions with ammonia or helium (Orlando et al. 1990a). We have also found that different bonds are fragmented in different ranges of kinetic energy
0
HZN-
Figure 13.
11 -C-
-OH
Six scissions of peptide bonds potentially generated by collisional activation.
Bieman 1988.
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YGLU-CYS-GLY
I
H-CYS-TYR-PHE-GLN-ASN-CYS-PRO-ARG-GLY-NHz
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0
200
yGLU-CYS-GLY
I
0
)
-
I000
0 00
600
400
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1400
b
H-CYS-TYR-PHE-GLN-ASN-CYS-PRO-ARG-GLY-NHz
1200
1400
M/Z - ... . . . . _. . . . .. . . . . .. . . Figure 14. Collisional activation spectra ot isomeric mixed disulphides tormed between glutathione and vasopressin.
(Orlando et al. 1990b). For example, in figure 16 the sticky complex formed between ammonia and N,N,N-triacetyl chitotriose is formed when the protonated trisaccharide is decelerated to provide collisions between 3 and 7eV, while cleavage of bonds adjacent to the amide group (which presumably is the site of the endothermic reaction with ammonia) occurs at energies between 10 and 14eV. At slightly higher kinetic energies (>15eV) ions are formed by cleavage in the carbohydrate linkages (Orlando et al. 1990 c). T h e potential application to glycopeptides and other glycosides is obvious.
Acknowledgements Research in the Structural Biochemistry Center at UMBC was supported by the National Science Foundation and the National Institutes of Health. H
I0 R-
Figure 15.
s
1
CHJS SIGH., - R '
Class characteristic cleavage in disulphide linkages.
C. Fenselau and P . B . W. Smith
1218 200
,
u
-
-
r
--n 0 C
1 204 (M+NH4lxlO NH4+x100
586(x50) 60(x50) 407 (x 1/21
Y
aH
ID
t
NH
NH
I
c:o
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I
0
10
20
30
40
I
c:o
c:o
CHI
CHI
I
50
E lab
Figure 16. Abundances of fragment ions formed by collisions between ammonia and N,N,N-triacetyl chitotriose at energies low enough to permit formation of complexes for endothermic reactions. Adapted from Orlando et al. 1990 c.
References BIEMANN, K., 1988,Contributions of mass spectrometry to peptide and protein structure. Biomedical and Environmental Mass Spectrometry, 16, 99-1 11. BUSCH,K.L., GLISH,G. L., and MCLUCKEY, S. A., 1988,Mass SpectrometrylMass Spectrometry (New York: VCH). M., Russo, J. E., HILTON,J., DULIK,D. M., and FENSELAU, C., 1988,Enzymatic mechanisms COLVIN, of resistance to alkylating agents in tumor cells and normal tissues. Adwances in Enzyme Regulation, 21, 21 1-221. COOKS,R. G. (ed.), 1978,Collision Spectroscopy (New York: Plenum Press). C., 1987,Conversion of melphalan to 4-(glutathionyl)-phenylalanine: a DULIK,D. M., and FENSELAU, novel mechanism for conjugation by glutathione-S-transferases. Drug Metabolism and Disposition, 15, 195-199. C., 1990, Characterization of glutathione conjugates of. DULIK,D., COLVIN,M., and FENSELAU, chlorambucil by fast atom bombardment and thermospray liquid chromatography/mass spectrometry. Biomedical and Environmental Mass Spectrometry, 19, 248-252. DULIK,D. M., FENSELAU, C., and HILTON,J., 1986. Characterization of melphalan-GSH adducts whose formation is catalyzed by glutathione transferase. Biochemical Pharmacology, 35,
3405-3410. FENSELAU, C . , and COTTER,R. J., 1987, Chemical aspects of fast atom mass spectrometry. Chemical Rwiews, 81, 501-512. FENSELAU, C., WANG,R., ODELL,G. B., and MOGILEVSKY, W., 1989,Plasma desorption characterization of glucuronide and glutathione conjugates derived from bilirubin. International Journal of Mass Spectrometry. Ion Processes, 92, 289-295. FENSELAU, C., YELLE, L., STOGNIEW, M., LIBERATO, D., LEHMAN, D., FENG,P., and COLVIN, M., 1983, Analysis of glucuronides by fast atom bombardment. International Journal of Mass Spectrometry und Ion Physics, 46, 41 1414. JOHNSON, R. S.,MARTIN, S. A., and BIEMANN, K., 1988,Collision-induced fragmentation of (M+H)* ions of peptides. Side chain specific sequence ions. fnternational Journal of Mass Spectrometry. Ion Processes, 86, 137-154. LINDHOLM, E.,1972,Mass spectra and appearance potentials studied by the use of charge exchange in a tandem mass spectrometer, in Ion-Molecule Reactions, vol. 2, edited by J. L. Franklin (New York: Plenum Press), pp. 457484. MCLAFFERTY, F. W. (ed.), 1983, Tandem-mass Spectrometry (New York: Wiley Interscience). ODELL,G . B., MOGILEVSKY, W. S., SMITH,P. B. W., and FENSELAU, C., 1991,The identification of glutathione conjugates of the dimethyl ester of bilirubin in the bile of Gunn rats. Molecular Pharmacology, 40, 597-605.
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1219
Downloaded by [Australian National University] at 05:18 07 May 2016
ORLANDO, R., FENSELAU, C., and COTTER,R. J., 1989, Endothermic ion molecule reactions: 11. Reactions between peptides with and without basic residues and ammonia. Organic Mass Spectrometry, 24, 1033-1042. ORLANDO, R., FENSELAU, C., and COTTER, R. J., 1990a, Endothermic ion-molecule reactions: 111. High energy collisional activation at low kinetic energies. Rapid Communications in Mass Spectrometry, 4, 259-262. ORLANDO, R., FENSELAU, C., and COTTER,R. J., 1990 b, Endothermic ion-molecule reactions. V. Remote-site fragmentation at very low kinetic energies. Organic Mass Spectrometry, 20, 485-489. ORLANDO, R., FENSELAU, C., and COTTER, R. J., 1990c, Endothermic ion-molecule reactions: IV. Sitedirected fragmentation in N-acetylated oligosaccharides at low beam energies. Analytical Chemistry, 62, 2388-2390. ORLANDO, R., MURPHY, C., FENSELAU, C., HANSEN, G., and COTTER, R. J., 1990d, Endothermic ionmolecule reactions: strategies for Tandem Mass Spectrometric structural analyses of large biomolecules. Analytical Chemistry, 62, 125-129. RICHTER, W. J., BLUM,W., SCHLUNEGGER, U. P., and SENN,M., 1983, Tandem mass spectrometry of pharmaceuticals, in Tandem Mass Spectrometry, edited by F. W. McLafferty (New York: Wiley Interscience), pp. 417-434. SHORE, L. S., MOGILEVSKY, W. S., and ODELL,G . B., 1989, T h e in oitro formation of glutathione conjugates of bilirubin dimethylester by hepatic microsomes of jaundiced Gunn rats. Hepatology, 10, 615(A). SHORE, L., MOGILEVSKY, W., SMITH, P., FENSELAU, C., and ODELL,G . , 1991, The in oitro formation of glutathione conjugates of the dimethylester of bilirubin. Biochemical Pharmacology, 42, 1969-1976. STRAUB, K. M., 1986, Metabolic mapping of drugs: rapid screening for polar metabolites using FAB MS/MS, in Mass Spectrometry in Biomedical Research, edited by S. Gaskill (Chichester: John Wiley and Sons). YUAN,2. M., SMITH, P. B., BRUNDRETT, R . B., COLVIN, M., and FENSELAU, C., 1991, Glutathione conjugation with phosphoramide mustard and cyclophosphamide: a mechanistic studying using tandem mass spectrometry. Drug Metabolism and Disposition, 19, 625-629.
