Differentiation of Isomeric N-Glycan Structures by Normal-Phase Liquid Chromatography−MALDI-TOF/TOF Tandem Mass Spectrometry

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 TOF/TOF-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.

High-Field Asymmetric-Waveform Ion Mobility Spectrometry and Electron Detachment Dissociation of Isobaric Mixtures of Glycosaminoglycans

“…Ion exchange methods such as high-performance anion-exchange chromatography (HPAEC) [36] have also been used for GAG separations. However, these present challenges when combined with MS analysis, requiring desalting or removal of ion pairing reagents prior to introduction to the mass spectrometer [37]. Capillary electrophoresis (CE) methods have also been used for separating and characterizing GAGs [31].…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for the period 2005–2006

“…Normal-phase HPLC coupled off-line to MALDI-TOF/TOF MS/MS has been reported to be a good method for isomer differentiation (Maslen et al, 2006). The TOF/TOF instrument produced abundant cross-ring fragment ions revealing linkage information.…”

Electron detachment dissociation of fluorescently labeled sialylated oligosaccharides

“…The primary amine of the label reacts with the aldehyde group of the glycan to generate a Schiff base intermediate, which is then stabilized through reduction to form a secondary amine [55]. 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminopyridine (2-AP), and 5-amino-2-naphthalenesulfonic acid (ANSA) are some of the commonly utilized fluorescent labels [56–68]. …”