Structural determination of neutral O-linked oligosaccharide alditols by negative ion LC-electrospray-MSⁿ.

Abstract

Neutral O-linked oligosaccharides released from the salivary mucin MUC5B were separated and detected by negative ion LC-MS and LC-MS 2 . The resolution of the chromatography and the information obtained from collision induced dissociation of detected [M Ϫ H] Ϫ ions were usually sufficient to identify the sequence of individual oligosaccharides, illustrated by the fact that 50 different oligosaccharides ranging from disaccharides to nonasaccharides could be assigned from the sample. Fragmentation was shown to yield mostly reducing end sequence fragments (Z i and Y i ), enabling primary sequence assignment. Specific fragmentation pathways or patterns were also detected giving specific linkage information. The reducing end core (Gal/GlcNAc␤1-3GalNAcol or Gal/GlcNAc␤1-3(GlcNAc␤1-6)GalNAcol) could be deduced from the pronounced glycosidic C-3 cleavage and A i type cleavages of the reducing end GalNAcol, together with the non reducing end fragment from the loss of a single substituted GalNAcol. Substitution patterns on GlcNAc residues were also found, indicative for C-4 substitution ( 0,2 A i Ϫ H 2 O cleavage) and disubstitution of C-3 and C-4 (Z i /Z i cleavages). This kind of fragmentation can be used for assigning the mode of chain elongation (Gal␤1-3/ 4GlcNAc␤1-) and identification of Lewis type antigens like Lewis a/x and Lewis b/y on O-linked oligosaccharides. In essence, negative ion LC-MS 2 was able to generate extensive data for understanding the overall glycosylation pattern of a sample, especially when only a limited amount of material is available. I dentification and understanding of biological processes involving glycoconjugates is a scientific area of increasing interest. This increase in interest has worked hand in hand with the development of more sensitive and specific analytical techniques in this field. Mass spectrometry has been the detection and characterization tool most frequently used in recent years for analysis of glycoproteins, glycopeptides, glycolipids, or free oligosaccharides (some reviews in [1][2][3][4][5]). In principle, mass spectrometry suffers from the fact that the monomeric components (the monosaccharides) of oligosaccharide chains consist of isomeric building blocks, and that linkage configuration and position are difficult to obtain with the technique. Even so, researchers have applied mass spectrometric techniques to answer biological questions (some examples in [6 -9]).Characterization of released or free oligosaccharides by mass spectrometry using electrospray ionization in negative mode has recently been shown to give both sequence and linkage information [10,11]. Negative ion mode has been shown to be applicable to both negatively charged oligosaccharides and for neutral oligosaccharides [12][13][14], and offers high sensitivity of detection without requiring derivatization or adduct formation to aid ionization. Electrospray is also easily coupled to with high resolution liquid chromatography, an important factor in resolving the degeneracy of structural isomers found i...

The influence of sialylation on glycan negative ion dissociation and energetics

“…The (1-3)-linked residues, or those with a blocked hydroxyl group in the 3-position, produce a C-type cleavage at the reducing side and a Z-type cleavage at the nonreducing side, a combination that is now termed D-type [10]. It is common to release O-linked glycans as reducing end alditols, and it has been demonstrated that abundant Z-type fragment ions are formed from dissociation of negatively charged precursor ions generated from asialo structures [25].…”

Human Synovial Lubricin Expresses Sialyl Lewis x Determinant and Has L-selectin Ligand Activity

“…The biosynthetic precursor of the non-sulfated core 2 structure with the composition Hex 2 HexNAc 2 ([M Ϫ H] Ϫ ions of m/z 749) was identified as a single isomer ( (Fig. 5A, inset) (50). Thus, this structure was annotated as the core 2 O-glycans, Gal␤1-4GlcNAc␤1-6(Gal␤1-3)GalNAc␣1- (50).…”

“…5A, inset) (50). Thus, this structure was annotated as the core 2 O-glycans, Gal␤1-4GlcNAc␤1-6(Gal␤1-3)GalNAc␣1- (50). Sulfated structures were also detected and sequenced after desialylation.…”

Thin chip microsprayer system coupled to quadrupole time-of-flight mass spectrometer for glycoconjugate analysis

“…In particular, in proteomics and glycoproteomics, nanoESI MS n in the positive as well as in the negative ion mode was shown to be capable of sequencing minute amounts of biological material thus providing straightforward information on various structure determinants. [1][2][3][4][5] Miniaturized analytical instrumentation is attracting growing interest in chemical, biochemical and structural analysis. Nowadays, various mass spectrometer configurations are interfaced to the integrated micro-and nanosystems or to devices providing either robotized sample infusion or a combination of automated sample delivery and chip-based ionization.…”