Glycan labeling strategies and their use in identification and quantification

Abstract

Most methods for the analysis of oligosaccharides from biological sources require a glycan derivatization step: glycans may be derivatized to introduce a chromophore or fluorophore, facilitating detection after chromatographic or electrophoretic separation. Derivatization can also be applied to link charged or hydrophobic groups at the reducing end to enhance glycan separation and mass-spectrometric detection. Moreover, derivatization steps such as permethylation aim at stabilizing sialic acid residues, enhancing mass-spectrometric sensitivity, and supporting detailed structural characterization by (tandem) mass spectrometry. Finally, many glycan labels serve as a linker for oligosaccharide attachment to surfaces or carrier proteins, thereby allowing interaction studies with carbohydrate-binding proteins. In this review, various aspects of glycan labeling, separation, and detection strategies are discussed.FigureMALDI-FTICR-MS of 2AA-labeled total plasma N-glycans

Structural Analysis of Variable Domain Glycosylation of Anti-Citrullinated Protein Antibodies in Rheumatoid Arthritis Reveals the Presence of Highly Sialylated Glycans

“…For each spectrum, a mass window of m/z 1000 to 4000 was used and a minimum of 5000 laser shots were accumulated. The identification of 2AA-labeled glycans by MALDI-TOF-MS was based on m/z values (only if the signal/noise Ͼ9) and literature data (supplemental Table S3) (22). The number of assigned and unassigned mass values above threshold of 9 within the linear negative mode spectra of AA-labeled glycan peaks, a median of 95% of all peaks in the intact IgG spectra, 88% of the peaks in the Fc spectra, and 94% of the peaks in the Fab spectra could be linked to glycan structures.…”

Evaporative fluorophore labeling of carbohydrates via reductive amination

“…Glycosylation is one of the major and most complicated forms of post-translational modifications that plays essential roles in the activity and effector function of monoclonal antibodies [3] demanding improved glycoanalytical methods. Frequently 3/13 used techniques for carbohydrate analysis are mass spectrometry (MS) [4], liquid chromatography (LC) [5,6], capillary electrophoresis (CE) [7][8][9], some combination of them [10][11][12] and if necessary, nuclear magnetic resonance spectroscopy (NMR) [13,14].…”

“…Procedures reported in the literature apply comparable but not unified reaction conditions and reagent volumes for fluorescent sugar labeling [10], most of them suggesting overnight incubation at 37°C [29,33] or several hours of reaction times at higher temperatures (50°C) [34].…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…A number of tags have been proposed for reductive amination; 66,86,87 some of these are shown in Fig. 4.…”