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Differentiation of Isomeric N-Glycan Structures by Normal-Phase Liquid Chromatography—MALDI-TOFI TOF Tandem Mass Spectrometry.

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Title: Differentiation of Isomeric N-Glycan Structures by Normal-Phase Liquid Chromatography—MALDI-TOFI TOF Tandem Mass Spectrometry.
Authors: Maslen, Sarah1, Sadowski, Pawel2, Adam, Alex3, Lilley, Kathryn2, Stephens, Elaine1 es287@cam.ac.uk.
Source: Analytical Chemistry. 12/15/2006, Vol. 78 Issue 24, p8491-8498. 8p. 1 Diagram, 7 Graphs.
Subjects: Glycosylation, Esterification, Spectrum analysis, Proteins, Nuclear isomers, Ionization (Atomic physics), Auger effect, Properties of matter, Phospholipases
Abstract: The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption! ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOP/TOP-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins. [ABSTRACT FROM AUTHOR]
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Database: Engineering Source
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Abstract:The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption! ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOP/TOP-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins. [ABSTRACT FROM AUTHOR]
ISSN:00032700
DOI:10.1021/ac0614137