Enhancing MS(n) mass spectrometry strategy for carbohydrate analysis: A b2 ion spectral library.

UNLABELLED

Searchable mass spectral libraries for glycans may be enhanced using a B2 ion library. Using a quadrupole ion-trap mass spectrometer, successive fragmentations of sodiated oligosaccharides were carried out in the positive ion mode. In B,Y-type fragmentation, disaccharide B2 ions are generated which correspond to specific glycosidic linkages using progressive MS stages. Fragmentation of "B2 ions" corresponding to glycosidic linkages such as Hex-Fuc, Hex-Hex, Hex-HexNAc, HexNAc-Hex and HexNAc-HexNAc, were systematically studied in low energy CID and collected to form a "B2 library". Linkages produce characteristic fragmentation patterns in the absence of cross-ring fragmentation. Patterns of "B2 ions" rely on relative stability of glycosidic bonds and carbohydrate-metal complexes in the gas phase. MS(n) studies of linear, branched trisaccharides and tetrasaccharides show that isomers for which B2 ion information is not available are rarely a problem in practice by their absence in an isomeric sequence or by their scarcity in nature. This MS strategy for linkage determination of carbohydrates aided by a "B2 library" was developed with a scope for expansion, providing an improved tool for glycomics. We validated this method examining levels of expressed activities of two glycosyl transferases in cancer cell lines: β3(B3GALNT2) and β4GalNAcT(B4GALNT3&4) that generate GalNAcβ3GlcNAcβ and GalNAcβ4GlcNAcβ.

BIOLOGICAL SIGNIFICANCE

Glycosylation is an important class of the "postranslationome", which includes manifold aspects of post-translational protein modification, affecting protein conformation, providing ligands for protein receptors [1-5], and encoding unique haptenic [6,7] or antigenic markers for oncology [8-11] and other applications. Identification of individual monomeric units, linkages, ring size, branching and anomerity has posed significant challenges to mass spectrometrists. MS(n) is a growing key instrumental method to differentiate among isomers [12]. While the potential isomers in oligosaccharides are impossibly large [12], likely possibilities can be limited by the biological system, including the expressed glycosyl transferases [13-20]. Mass spectra from sequential stages of collision activation (MS(n)) can supply structural details for precise characterization of linkage, monomer ID, substitutions, anomerity and branching [21-25]. There is a fundamental need for high throughput tools in glycomics to complement proteome studies. In that regard, nothing could be more important than searchable spectral library files for structural confirmation. The National Academy of Science (NAS) report (http://glyco.nas.edu) recommends the need of more than 10,000 synthetic structures of carbohydrates to advance the field of glycomics. This study demonstrates that the general reproducibility of ion trap spectra, and energy independence from modes of ionization and collisional activation, make compiling an MS(n) library for carbohydrate identification an achievable research target [26]. We intend to use the new B2 library for carbohydrate differences found on cancers, where we profile the glycosyltransferases to predict classes of potential structures, and use the library for MS identification of the expected cohort of altered structures.