Anal. Chem. 1001, 63,1463-1466
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Analysis of Neutral Oligosaccharides by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Bernd Stahl and Martin Steup Znstitut fur Botanik, Uniuersitat Munster, Schlossgarten 3, 0 - 4 4 0 0 Munster, Federal Republic of Germany Michael Karas* and Franz Hillenkamp Institut fur Medizinische Physik, Uniuersitat Munster, Robert-Koch-Strasse 31, 0-4400 Munster, Federal Republic of Germany
MaMx-aesleted laser desorption lonizatbn mass spectrometry (LDI-MS) was used for mass determination of neutral nonderivatlzed oligosaccharides and mixtures thereof. DIhydroxybenzoic acid or caffelc acid served as matrix. A nitrogen laser (A = 337 nm, 7 = 5 ns) was used for Ion demrptlon. For comparison digogiucans were analyzed by both high-porformance and thin-layer chromatography. With LDI-MS glucan species were generally detected as monosodium and monopotassium carbohydrate adduct lons. Addition of RMum chbrlde to the analyte resuited in the formation of an additional monolithium glucan adduct ion. No fragmentatlon of the analyte was observed. LDI-MS as described here was also applled to analyze oligofructans, mannose-contalnlngoligosaccharides and glycoliplds. LDIMS has the advantages of being able to resolve complex carbohydrate mixtures, of high sensitivity and simplicity of analyte preparation; sample pretreatment, such as desalting is usually not required for LDI-MS.
INTRODUCTION Oligosaccharides represent a heterogeneous group of biologically important compounds, either as free carbohydrates or as constituents of glycoconjugates. Free oligosaccharides occur as intermediates or end products of the cellular carbon metabolism. In plants they play an important role in carbon fluxes within the cells and in the entire organism (I, 2). Oligosaccharides covalentlybound to non-glycan constituents (such as proteins), are involved in a variety of biological functions (3-5). Both free and bound oligosaccharides can occur as mixtures of oligomers that differ in features, such as degree of polymerization, monomer composition, and the type(s) of intersugar linkage(8 ) . Therefore, biochemical analysis of oligosaccharides often requires both the resolution of complex carbohydrate mixtures and the characterization of the individual oligomer type. Oligosaccharide mixtures can be separated by a variety of established methods, among which are gel filtration (6, 7), thin-layer chromatography (8-101, and high-performance liquid chromatography (11, 12). These techniques are of limited use in accurate mass determination. Oligosaccharides having the same degree of polymerization but differing in molecular shape (e.g. linear or cyclic molecules) often possess different chromatographic characteristics. Thus a correlation between retention time and size of an oligosaccharide holds only within a homologeous series of oligomers. The use of structurally identical molecular weight (MW) standards is essential. Therefore, molecular weight determinations of compounds of unknown structure and of mixtures thereof are difficult to perform. 0003-2700/9 1/0363-1463$02.50/0
Mass spectrometric techniques such as fast atom bombardment have been applied both for molecular weight determination and analysis of glycans up to a MW of 3500 (13-1 7). However, for neutral underivatized oligosaccharides FAB exhibits a low ion yield and thus sensitivity (1-50 nmol of sample required (17)). Derivatization is typically used to either enhance surface activity or introduce preferred sites for (de)protonation. In this communication results of matrix-assisted UV laser desorption/ionization mass spectrometry (LDI-MS) of native neutral homoglycans are presented. The method, recently developed by some of the authors, has until now mainly been used for molecular weight determination of proteins up to masses of ca. 350000 Da (18-22). As shown in this report, LDI-MS is also a powerful tool for the determination of molecular masses and size distribution of oligosaccharides. In a first experiment a linear and a cyclic crglucan, maltoheptaoseand cycloheptacee, respectively, were analyzed separately and as a mixture. LDI-MS has then been used for analysis and mass determination of commercial maltodextrins and dextrans. Finally, the technique has been applied to the evaluation of the kinetics of an enzymatic polymerization reaction. For this purpose, glucosyl transfer from glucose 1-phosphate to a primer, catalyzed by glucan phosphorylase (E.C.2.4.1.1.), was chosen.
EXPERIMENTAL SECTION LDI-MS. The mass spectrometer used was a reflector type time of flight (TOF) microprobe instrument (LAMMA 1ooO) with a nitrogen laser (Laser Science Inc.) of 337-nm wavelength and 5-ns pulse width. The apparatus has been described in detail elsewhere (23).The laser beam was focused onto the sample at an angle of 4 5 O to the surface normal. Typical spot sizes range from 10-30 pm, with irradiances at the sample surface of 106-10' W/cm2. Ions were accelerated to an energy of 3 keV before entering the TOF mass spectrometer. At the detector ions were ptaccelerated to a maximum kinetic energy of up to 20 keV for more efficient detection by a secondary electron multiplier (EM1 9643). The analogue signals were recorded with a transient digitizer (LeCroy 9400) with 10 or 20 ns/channel resolution and then transferred to a PC. All spectra shown are accumulations of 10-60 single spectra. Measurements typically take 10-15 min, including sample preparation. Analyte Preparation for Mass Spectrometric Investigation. 2,B-Dihydroxybenzoic acid was used as matrix except when stated otherwise. It was dissolved to a concentrationof 10 g/L in a 10% (v/v) ethanol-water solution. Analytm (0.1-1 g/L) were prepared in doubly distilled water and then diluted with an appropriate volume of the matrix solution (typically 1:4 to l:lO, v/v). Aliquots of the resulting mixture (1pL or less) were placed on a piece of silver plate. The solvent was removed in a gentle stream of air and the solid sample/matrix mixture was then transferred into the vacuum chamber of the mass spectrometer. In some experiments dihydroxybenzoic acid was replaced by derivatives of cinnamic acid (e.g. caffeic or ferulic acid). In these 0 1991 American Chemlcal Society
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ANALYTICAL CHEMISTRY, VOL. 63, NO. 14, JULY 15, 1991
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Flgurs 1. Matrix LDI mass spectra of cycloheptaose(A, B), makoheptaose (C), and an equlmolar mixture of both heptaoses (D): (A) complete spectrum: (B-D) expanded analyte ion mass region: index “c” and “m” for cycloheptaose and maltoheptaose, respectively. Conditions: matrix, dlhydroxybenzoicacid; sample, 50 ng of heptaose (A-C), 100 ng (D). The spectra are averages of 10 single spectra each.
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Flgure 2. Matrix LDI spectra of cycioheptaose (A, B) and maltoheptaose (C, D): (A, C) llthiumfree sample; (B, D) sample In 0.16 mM
LICI. Conditions: matrix, dihydroxybencoic acid. The spectra are averages of 20 spectra: index “c” and “m” for cyclohepteose and maltoheptaose,respectively. 4.
