Analysis of glycoprotein oligosaccharides by fast atom bombardment mass spectrometry.
ABSTRACT A strategy is outlined for isolation and structural analysis of oligosaccharides derived from glycoproteins. Oligosaccharides, N- and O-linked, are released by chemical and enzymatic methods and separated using gel filtration, concanavalin A affinity chromatography and high-performance ion-exchange chromatography. Structural studies are carried out by fast atom bombardment mass spectrometry. The structural information from the latter can be extended to include determination of binding positions between monosaccharide residues. Prior to the analysis, samples are subjected to periodate oxidation, reduction and permethylation. The positions of glycosidic linkages are deduced from the spectrum by the primary and secondary sequence ions. Structural information can be obtained from mixtures of isomeric compounds.
Glycoconjugate Journal 08/1993; 10(4):335-335. DOI:10.1007/BF01210158 · 1.95 Impact Factor
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ABSTRACT: The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N-glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N-linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N-glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N-glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.Journal of the American Society for Mass Spectrometry 09/2012; 23(11):1955-66. DOI:10.1007/s13361-012-0425-8 · 3.59 Impact Factor