Anal. Chem. 1985, 57, 2287-2289
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Stereoisomer Differentiation of 2-Acetamido-2-deoxyhexose by Mass Spectrometric Measurement of Relative Gas-Phase Alkali Ion Affinities Jean-Jacques Fourniii and Germain Puzo*
Centre de Recherche de Biochimie et de GEngtique Cellulaires du C.N.R.S., 118, route de Narbonne, 31062 Toulouse Cedex, France
Catlonlzed dlmers [hexosamlne-cat-matrlx]' of 2-acetamldo-2-deoxy-~glucose,2-acetamldo-2-deoxy-~-mannose, and 2-acetamldo-2deoxy~alactosewere generated by fast atom bombardment (FAB) uslng different alkall catlons and glycerol, S-valerolactame, and diethanolamine matrlces. From thelr unimolecular decomposltlon (metastable Ions) malnly two klnds of Ions are observed resultlng from the retention of the cation either by the amino sugar (Ion a) orland by the matrlx (Ion b). For a given hexosamlne their relatlve abundance depends on the alkali cation and the matrlx used. When the matrlx Is diethanolamine and the alkall catlon Is ltthium or sodlum, the N-acetylglucosamine stereolsomers can be dlfferentlated by the relatlve abundances of ions a and b of the catlonlzed dimer.
Amino sugars are important constituents of antibiotics such as aminoglycosides ( I ) , biological polymers such as glycoproteins (2), and lipopolysaccharides (3) which can present immunological activities. Among these amino sugars the N-acetylhexosamines have been extensively studied by mass spectrometry. Their identification requires great mass spectrum selectivity due to their isomeric forms. Structural investigation by EI/MS and CI/MS involves their derivatization. E1 fragmentation pathways of MeaSi ethers of 2-acetamido-2-deoxyhexosehave already been established. Differences between mass spectra are insufficient to allow anomer or epimer identification ( 4 ) . However it has been shown that EI-MS of permethylated N-acetylhexosamines allows the localization of the acetamido group in the C-1, (2-2, (2-3, or C-4 positions. Moreover, CI of MesSi derivatives of some 2-N-acetyl hexosamine stereoisomers using methane as reactant gas gives mass spectra in which it is the relative intensities of the fragment ions that allows their differentiation (5). Recently, the use of surface ionization modes such as FD, laser, and FAB has permitted the structural analysis of underivatized oligosaccharides (6). Among these different ionization modes FAB seems the most suitable since molecular weight and sequencing information have been obtaind from their mass spectra in both the positive and negative mode (7, 8). FAB ionization requires the use of a matrix, usually glycerol, in which the sample is dissolved (9). We have shown by MIKE analyses that one way to generate protonated or cationized disaccharide molecular ions is to desolvatate protonated or cationized dimers (see Scheme I) (10, 11). Similar cationized dimers [S-cat-M]+ are generated by FAB from aldohexoses and methyl glycosides (12-14). Moreover as described in Scheme I it has been observed that their unimolecular dissociation gives two kinds of ions: a and b, whose relative abundances allow epimer and anomer differentiation of certain glycosides (12,14). Using a similar method Cooks et al. (15) ordered the relative gas phase (Ag+)affinities 0003-2700/85/0357-2287$01.50/0
S I sugar analyzed cat: alkali ion M, matrix
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
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E / E, Flgure 1. MIKE spectrum of sodium-bound dlmer [(NAcGlc)-Na-
glycerol] +.
P
.
I (NAcGls N t O E A l 21
xm
mam beam
Flgure 3. Concentration effect of NAcGlc at the target surface on the relative abundances of metastable Ions a and b arising from the cationized dimer [(NAcGlc)-Li-DEA]'.
I'1
intensit ratio a J
Table I. a/b Ratios of the Abundance of Metastable Ions a and b Arising from the Cationized Dimer [ (2-NAcGlc)-cat+-DEA] for cat = Li+, N+, K+, Rb+ a/ b ratios
alkali cation
Li
Na
K
Rb llme(s,.c)
[(2-NAcGlc)-cat+-
0.36 i 0.05 1.3 f 0.2 11 rt 3 330 f 80
DEAl sponding to the retention of the sodium cation by NAcGlc. Rather than increasing the internal energy of the precursor ion (cationized dimer) by collision activation for ion b [glycerol-Na]+ formation, we selected a higher basicity matrix, DEA. Indeed, it has been observed that the reproducibility of MIKE spectra of bound dimer is better with ions of low internal energy (metastable ions) (16). Figure 2 shows the MIKE spectrum of the sodium-bound dimer [NAcGlc)-NaDEAI+in which DEA replaces glycerol. Two signals of similar magnitude observed corresponding to ions a and b are [ (NAcG1c)-Na]+ and [DEA-Na]+, respectively. So a DEA matrix was used to distinguish the hexosamine stereoisomers. Alkali Ion Effects on Cationized Dimer Formation and on Its Unimolecular Decomposition. With the aim of producing cationized dimers, we doped a solution of NAcGlc in DEA with an equimolecular mixture of the different alkali cations in the form of their iodine salts so that each cation was in a ratio of one to ten molecules of NAcGlc. In the high mass range all the cationized molecular ions [(NAcGlc) + cat]' and cationized dimers expected with the different alkali cations used were observed. We were therefore able to record the MIKE spectra of each cationized dimer except for the cesium-bound dimer whose abundance was too low. Table I summarizes the intensity ratios between ions a and b. An increase in the a/b ratio is observed with the rise in the alkali cation radius. For instance, this ratio increases by a factor of about 1000 when rubidium is present in the cationized dimer instead of lithium. From these results lithium- and sodium-bound dimers, which give approximately the same amount of metastable ions a and b, seem the most suitable for hexosamine stereoisomer differentiation. Source Parameters and Concentration Effects on the Ratio a/b. MIKE spectra allow the characterization of ions
ANALYTICAL CHEMISTRY, VOL. 57, NO. 12, OCTOBER 1985
Table 11. a/b Ratio of the Relative Abundances of Metastable Ions a and b (MIKE) Resulting from the Different Cationized Dimers a l b ratio cationized dimer
[ (2-NAcGlc)-cat-DEA] [ (2-NAcGal)-cat-DEA]+ [ (2-NAcMan)-cat-DEA]+
+
Li
Na
0.36 f 0.05' 4.3 f 0.7 18 5
1.3 f 0.2 41 f 6 9.7 f 0.6
-
-
Error corresaondine t o the intensitv rearoducibilitv of aeaks a a n d b during two cons&utive scans of -the ESA.
10 s after the introduction of the probe in the FAB source and the starting of the atom gun. Figure 4 shows that the a/b ratio remains almost the same (0.43 f 0.03) in the five MIKE spectra recorded between tdOand tsW and increases after 4 min. This phenomenon could result for instance, from a modification of the amino sugar structure at the probe tip. It is well-known that in aqueous solutions the aminohexoses investigated are mainly present in equilibrium under two pyranoside forms, cy and p (18). The modification of their proportion and the appearance of other forms, such as furanose, has been observed in the presence of high concentrations of inorganic salts. We can assume that similar situations can occur at the probe tip, and an increase of the salt concentration due to the higher volatility of the matrix could modify the proportion of the forms and the structure of the aminohexose investigated. Also since the temperature at the target surface is not controlled, its increase could involve the modification of the equilibrium between the amino sugar structures which may explain the variation of the a/b ratio. By methylation of the hydroxyl group linked to the anomer carbon, this equilibrium is blocked and the examination of the decomposition of their cationized dimers could give a fuller understanding of the phenomenon previously described. Similar effects have not been observed when methyl glycosides are analyzed (14). MIKE Analysis of Cationized Dimers of Hexosamine Stereoisomer. The optimization of generating cationized dimer and giving reproducible a / b ratios takes into account the previously described parameters. The cationized dimers from compounds 1, 2, and 3 were generated by FAB in the presence of DEA as matrix and sodium or lithium iodide as alkali salt. The relative abundances of the ions a and b resulting from the unimolecular decomposition (MIKE analysis) of the different cationized
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dimers investigated is given in the Table 11. We observe that the decomposition of both sodium- and lithium-bound dimers is characteristic of the stereoisomer involved in the dimer. For instance, if we consider lithium-bond dimer dissociation, we observe that the a/b intensity ratio is 50 times higher for the dissociation of [ (NAcMan)-Li-DEAI+ than for [ (NAcG1c)Li-DEAI+. It can also be seen, from Table 11, that the classification of lithium affinity of the hexosamine stereoisomers 1,2, and 3-NAcMan > NAcGal > NAcGlc-does not hold for sodium since an inversion occurs between NAcMan and NacGal to give the following sequence NAcGal > NAcMan > NAcGlc. A similar sequence is observed for gas phase ion (K+)affinity. This phenomenon is not yet very clear, and its understanding would involve the determination of the cationized dimer conformations. ACKNOWLEDGMENT We wish to thank Professor Winterton for rereading the manuscript. Registry No. 3,1811-31-0;DEA, 111-42-2; Li, 7439-93-2;Na, 7440-23-5; K, 7440-09-7;Pb, 7440-17-7; glycerol, 56-81-5; 6-Valerolactam, 675-20-7. LITERATURE CITED (1) Asselineau, J.; Zalta, J. P. "Antlblotiques"; Hermann: Paris, 1973. (2) Lennarz, W. J., Ed. "Biochemistry of Glycoproteins and Proteoglycans"; New York, 1980. (3) Asselineau, J. "The Bacterial Llplds"; Lederer, E., Ed.; Hermann: ParIs, 1966. (4) Waller, G. R., Ed. "Biochemical Applications of Mass Spectrometry"; Wlley-Interscience: New York, 1972. (5) Bowser, D. V.; Teece. R. G.; Somanl, S.M. Biomed. Mass Spectrom. 1978, 5 , 627-633. (6) Reinhold, V. N.; Carr, S. A. Mass Spectrom. Rev. 1983, 2 , 153-221. (7) Dell, A.; Ballou, C. E. Blomed. Mass Spectrom. 1983, 10, 50-56. (8) Dell, A.; Ballou, C. E. Carbohydr. Res. 1983, 120, 95-111. (9) Barber, M.; Bordoli, R. S.: Sedwlck, R. D.; Tyler, A. N. J. Chem. SOC., Chem. Commun. W81, 325-326. (10) Puzo, G.; Prom& J. C. Org. Mass Spectrom. 1984, 19, 448-451. (11) Puzo, G.; Prome, J. C. Org. Mass Spectrom. 1985, 2 0 , 288-291. (12) Puzo, G.; Prom6, J. C. Spectrosc.: Int. J. 1984, 3 , 155-158. (13) Puzo, G.; Fournis, J. J.; Proms, J. C. Anal. Chem. 1985, 5 7 , 842-844. (14) Puzo, G.; Prom& J. C.; Fourni6, J. J. Carbohydr. Res., in press. (15) McLuckey, S. A.; Shoen, A. E.: Cooks, R. G. J. Am. Chem. SOC. 1982, 104, 848-850. (16) McLuckey, S. A.; Cooks, R. G. Int. J. Mass Spectrom. Ion Phys. 1983, 5 2 , 165-174. (17) Cooks, R. G.; Beynon, I . H.; Caprioli, R. M.; Lestezr, G. R. "Metastable Ions"; Elsevler: New York, 1973. (18) Angyal, S. J. Adv. Carbohydr. Chem. Biochem. 1984, 42, 15-62.
RECEIVED for review February 27, 1985. Accepted May 28, 1985.
