Structural Documentation of Glycan Epitopes: Sequential Mass Spectrometry and Spectral Matching

Abstract

Documenting mass spectral data is a fundamental aspect of accepted protocols. In this report we contrast MSn sequential disassembly spectra obtained from natural and synthetic glycan epitopes. The epitopes considered are clusters found on conjugate termini of lipids and N-, and O-glycans of proteins. The latter are most frequently pendant through a CID-labile HexNAc glycosidic linkage. The synthetic samples were supplied by collaborating colleagues and commercial sources and usually possessed a readily released reducing-end linker, a by-product of synthesis. All samples were comparably methylated, extracted, and MSn disassembled to compare their linkage and branching spectral details. Both sample types provide B-ion type fragments early in a disassembly pathway and their compositions are a suggestion of structure. Further steps of disassembly are necessary to confirm the details of linkage and branching. Included in this study were various Lewis and H antigens, 3- and 6-linked sialyl-lactosamine, NeuAc-2,8-NeuAc dimer, and Galα1,3Gal. Sample infusion provided high quality spectral data while disassembly to small fragments generates reproducible high signal/noise spectra for spectral matching. All samples were analyzed as sodium adducted positive ions. This study includes comparability statistics and evaluations on several mass spectrometers.

Structural Characterization by Multistage Mass Spectrometry (MSn) of Human Milk Glycans Recognized by Human Rotaviruses

“…For internal Fuc-LacNAc units, spectrum matching with standards has indicated that these structures match very well with the B/Y-type Fuc-LacNAc trisaccharide fragment isolated for sialylated Lewis X standards. This sim- ilarity essentially indicates that this unit is an internal Lewis X structure, and the m/z 646 fragmentation matches that standard trisaccharide spectrum (12). The isomeric mixture spectrum isolated from HMG-51 is shown in Fig.…”

“…Fucose localization, branching/ topology, linkages, and other epitope structure assignments are similar for all (23).…”

“…Data interpretation involved a combination of de novo and library-matching approaches. The evaluation of oligosaccharide standards, reproducibility, and comparison with biological samples is well appreciated (12). Where indicated, CID spectra of HMG fragment spectra were compared with those of standards for identification.…”

“…Furthermore, the intensity pattern of the internal LacNAc is indicative of the -OH group at the 3-position of the galactose, rather than the 6-position, thus the assignment of the three-linkage between the LacNAc units. This last assignment was based on spectral comparisons with standard materials and was supported by computationally assigned spectral-matching scores (supplemental material HMG-13) (23).…”

“…To approach such detail, we describe here a stepwise disassembly to obtain low molecular weight ion fragments where structural detail is exposed. Such fragment spectra are matched and compiled in a library that has been documented with synthetic standards (12).…”