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Field Desorption Mass Spectrometry of Oligosaccharides

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Field Desorption Mass Spectrometry of Oligosaccharides Proc. Nati. Acad. Sci. USA Vol. 78, No. 3, pp. 1471-1475, March 1981 Biochemistry Field desorption mass spectrometry of oligosaccharides (methylmannose polysaccharide/glycoprotein core oligosaccharide/molecular weight/sequence determination) MICHAEL LINSCHEID*t, JAY D'ANGONA*t, ALMA L. BURLINGAME*t, ANNE DELLS, AND CLINTON E. BALLOU§ *Space Sciences Laboratorv, University of California, Berkeley, California 94720; tDepartment of Pharmaceutical Chemistry, University of California, San Francisco, California 94143; tDepartment of Biochemistry Imperial College of Science and Technology, London, England; and §Department of Biochemistry, University of California, Berkeley, California 94720 Contributed by Clinton E. Ballou, November 21, 1980 ABSTRACT Field desorption mass spectrometry has been steps, and interfere with the ability to distinguish between used to analyze carbohydrate polymers with 5 to 14 hexose units methylated and unmethylated hexoses. In this report, we de- without prior derivatization. In all examples, the molecular weight of the oligosaccharide could be determined by means of the abun- scribe the application of field desorption mass spectrometry dant %uasimolecular ions of the type MNa+, MH+, MNa22+, and (FDMS) for analysis of underivatized oligosaccharides with up MNa3 +. Fragmentation at glycosidic linkages was observed in to 14 hexose units. We outline the limitations imposed by mass varying extents. The reduced oligosaccharide Man8GlcNAcH2, spectrometers of mass range 1000 and show how these limita- obtained from IgM [Cohen, R. E. & Ballou, C. E. (1980) Bio- tions can be partly overcome by summation of data. Further- chemistry 19, 4345-4358], gave quasimolecular ion signals MNa+ more, we demonstrate the utility of the high-field magnet mass at m/z 1544, MH+ at m/z 1522, MNa22+ at m/z 784, and MNa33+ at m/z 530, all corresponding to its assumed molecular weight of spectrometer (7) in FDMS studies of high mass compounds. 1519.5. Mycobacterial methylmannose polysaccharides with the Although FDMS suffers the disadvantage of nonreproduc- general structure ManxMeMany-OCH3 [Yamada, H., Cohen, R. ible signal intensities due to strongly fluctuating ion currents, E. & Ballou, C. E. (1979) I Biol. Chem 254, 1972-1979] were also and the instrument characteristics such as emitter quality, ion successfully analyzed Man MeMan13-OCH, the largest homo- source, and instrument performance must be evaluated em- log, gave the expected signal of the quasimo ecular ion MNa' at pirically (8), the success in structure analysis of small oligosac- m/z 2506. The larger polysaccharides were analyzed by using a KRATOS MS-50 mass spectrometer with a high-field magnet en- charides and glycosides in ranges up to -1300 mass units was abling full sensitivity to be maintained up to 3000 atomic mass very encouraging (9, **). These experiments suggested that one units. Polysaccharides up to m/z 1978 were analyzed by using a could expect strong quasimolecular ions and limited fragmen- KRATOS MS-9 mass spectrometer operated at 4 Kv. The signal- tation at the glycosidic linkages. to-noise ratio, which becomes a serious problem in field desorption Here we show that the ion MNa' for a high-mannose oli- mass spectrometry at low accelerating voltages, and the low in- gosaccharide (Man8GlcNAcH2) of the type found in immuno- strument sensitivity were improved considerably by our use of a method of adding scans with low total ion currents obtained over globulins (10, 11) is readily obtained at m/z 1544. We have also a longer desorption time. In this way, we obtained complete se- analyzed the mycobacterial methylmannose polysaccharides quence information on methylmannose polysaccharides up to (12) and have observed the quasimolecular ion MNa+ for the ManMeMang-OCH3 (MNa+ at m/z 1802). Analysis of a presumed largest homolog (ManjMeMan13-OCH3) at m/z 2506, whereas Man1MeMan7-OCH3, gave a spectrum consistent only with the for shorter homologs we have obtained fragmentation patterns structure Man2MeMan6-OCH3, revealing the existence of a meth- that allow determination of the complete sequence of the mol- ylmannose homolog with 2 unnmethylated mannoses at the non- ecules. A preliminary report of some of these results has been reducing end of the chain. published (13). The determination of exact molecular weights for carbohydrate MATERIALS AND METHODS polymers with 10 to 20 sugar units has been impossible because available Materials. The 3-0-methylmannose polysaccharides were methods (sedimentation, reducing-end determination isolated from Mycobacterium smegmatis (14) and were frac- by oxidation or reduction, periodate oxidation, and gel filtra- tionated and purified by high-performance liquid chromatog- tion) had inherent errors of 5% or more. Recentlv, electron im- on a pact mass spectrometry has been used with success for structur- raphy Waters Associates ,Bondapack/carbohydrate col- al umn (elution with acetonitrile/water) and by gel filtration on analysis of oligosaccharides and glycosides, and derivatiza- a Bio-Gel P-4 column (elution with water) (12, 15). Two ho- tion methods to make the material sufficiently volatile (silylation, mologous series of compounds have been obtained, one com- acetylation, and methylation) have been studied in detail (1, 2). posed entirely of al-s'4-linked 3-O-methylmannose and the Although intensities of significant ions decrease strongly with other terminated at the nonreducing end by a single al-*4- increasing molecular weights, spectra of these derivatives have linked mannose. Both series are terminated at the reducing end allowed sequence analysis of oligosaccharides (3, ¶), detection by a methyl aglycone. of branching (11), and assignment of attachment sites of agly- cones in complex lipids (4, 5) and conjugates (1, 6). The IgM oligosaccharide Man8GlcNAc was isolated after en- Our present objective is to develop, in the range 1500 to 3500 doglucosaminidase H digestion of a glycopeptide from an im- mass units, a method suitable for studies of carbohydrate chains munoglobulin fraction obtained from a patient with Walden- with 10 or more hexose units. We have chosen to avoid deri- vatization methods because they increase the molecular weight, Abbreviation: FDMS, field desorption mass spectrometry. ¶ Leffler, H. (1979) 27th International Union of Pure and Applied consume additional sample during the necessary purification Chemistry Conference, Helsinki, Finland. II Hansson, G. C. (1979) 27th International Union of Pure and Applied The publication costs of this article were defrayed in part by page charge Chemistry Conference, Helsinki, Finland. payment. This article must therefore be hereby marked "advertise- ** Burlingame, A. L. & Kambara, H. (1979) 27th International Union ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. of Pure and Applied Chemistry Conference, Helsinki, Finland. 1471 Downloaded by guest on September 26, 2021 1472 Biochemistry: Linscheid et at Proc. Nad Acad. Sci. USA 78 (1981) A 1522 1219 1381 1359 iii L id 1 I 11 II I B 1544 211 fI i I 11 I , I A 530 I 21iJll b lii L B 1784 II It II ". III, 11 II1 oI II i I1AId FIG. 1. FDMS of Man8GlcNAcH2. Partial galvanometer records for a scan from m/z 1600 to 500 at 30 sec/decade. Tracings A and B are con- secutive scans. The ion assignments are given in the text. strom's macroglobulinemia (10) and was reduced with sodium KRATOS); 8 kV accelerating voltage was used and spectra, re- borohydride to give Man8GlcNAcH2 (calculated molecular corded with a UV trace strip-chart recorder, were calibrated weight, 1519.5). The molecular size was determined by gel fil- with a crystal time marker by comparison with the electron-im- tration on a Bio-Gel P-4 (-400 mesh) column that had been pact spectrum of Fomblin oil. The scan speed was 30 sec/de- calibrated with a series of related oligosaccharides, and the size cade and the appropriate mass range to detect the quasimole- was confirmed by integration of the anomeric proton NMR cular ions, scan length between 200 and 900 mass units, was signals relative to the one (-linked mannose unit in the covered. Samples were loaded by dipping the emitter wire into oligosaccharide. 2-5 dul of a methanol solution of the oligosaccharide (1-5 /Lg/ Methods. The mass spectra were recorded by two methods. ml). The IgM oligosaccharide was scanned from m/z 1700 to m/z The spectra of the IgM oligosaccharide and of the larger meth- 400 in order to include fragment ions. The high-field magnet ylmannose polysaccharides were obtained with a MS-50 mass was scanned repetitively while the emitter current was slowly spectrometer (KRATOS, Manchester, England) at the Depart- increased. When the total ion current monitor indicated that ment of Biochemistry, Imperial College, London. The instru- desorption was occurring, the UV monitor was triggered. Sev- ment is equipped with a combined electron impact-field de- 'eral scans, all with excellent signal-to-noise ratios, could be ob- sorption source and a high-field magnet (developed by H. R. tained from each emitter loading, and with the IgM sample the Morris, Imperial College, in collaboration with S. Evans of ion beams lasted for about 1 min. 736 MNo+2X 498 1450 MNo| .583 231 407 752883 126 111,2 11288 .. ... ... X 250 500 750 1000 1250 1500 FIG. 2. (Lower) FDMS of ManMeMan7-OCH3: results of summation of five scans. (Upper) How the polysaccharide is assumed to fragment to give the observed ions. Only reproducible ions without isotope signals are displayed. Downloaded by guest on September 26, 2021 Biochemistry: Linscheid et al. Proc. Nati. Acad. Sci. USA 78 (1981) 1473 Data on the smaller methylmannose homologs were acquired Table 1. Molecular weights and quasimolecular ions of the with the MS-9 mass spectrometer (KRATOS) also equipped methylmannose polysaccharide homologs with a combined electron impact-field desorption source and Calculated connected to the Berkeley LOGOS II Datasystem (Mass Spec- Postulated molecular Quasimolecularions trometry Resource, Space Sciences Laboratory, University of structure weight MNa' MNa22+ MNa33+ California, Berkeley) (16).
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