Identification of isomeric N-glycan structures by mass spectrometry with 157 nm laser-induced photofragmentation

Abstract

Characterization of structural isomers has become increasingly important and extremely challenging in glycobiology. This communication demonstrates the capability of ion-trap mass spectrometry in conjunction with 157 nm photofragmentation to identify different structural isomers of permethylated N-glycans derived from ovalbumin without chromatographic separation. The results are compared with collision-induced dissociation (CID) experiments. Photodissociation generates extensive cross-ring fragment ions as well as diagnostic glycosidic product ions that are not usually observed in CID MS/MS experiments. The detection of these product ions aids in characterizing indigenous glycan isomers. The ion trap facilitates MS n experiments on the diagnostic glycosidic fragments and cross-ring product ions generated through photofragmentation, thus allowing unambiguous assignment of all of the isomeric structures associated with the model glycoprotein used in this study. Photofragmentation is demonstrated to be a powerful technique for the structural characterization of glycans. , and protein regulations and interactions [6], is widely acknowledged [7]. To improve our understanding of these processes, it is important to explore glycan structure-function relationships. However, the detailed characterization of a glycan structure and its attributes remains a difficult task, given the microheterogeneity and diversity of these molecules. Importantly, the discrimination of numerous structural isomers differing in sequence, linkage, position, or branching features remains a challenging frontier of glycobiology.For years, the characterization of glycan structures has almost exclusively been accomplished by tandem mass spectrometry (MS/MS) [1,8,9] because of its high sensitivity and minimum sample requirements relative to nuclear magnetic resonance (NMR). Different combinations of ionization techniques, ion activation, and A glycan ion generally fragments in two ways: (1) glycosidic cleavages resulting from a bond rupture between two adjacent sugar residues and (2) cross-ring cleavages in which any two bonds on the same sugar unit are broken. Cross-ring fragment ions are commonly observed in high-energy collision-induced dissociation (CID) methods as demonstrated in the tandem TOF/TOF approach [34 -38]. A limitation of this technique, however, is its inability to perform multistage tandem mass spectrometry experiments. On the other hand, glycosidic cleavage ions are predominantly observed in low-energy activation methods and are mainly used to derive sequence and limited branching information. Most recently, demonstrated the use of low-energy activation to identify the structural isomers of glycans in complex mixtures with the help of sequential tandem mass spectrometry (MS n ). However, a disadvantage of CID is the decrease in both the degree and efficiency of dissociation with increasing mass and MS n events. Alternatively, other activation techniques have been used for Address reprint requests to Prof. James P.

Wavelength-tunable ultraviolet photodissociation (UVPD) of heparin-derived disaccharides in a linear ion trap

“…Whereas high photon energy [20,22] enables the photofragmentation of native oligosaccharides, derivatization with a fluorophore is used for visible and near UV photofragmentation of oligosaccharides [21,23]. To delineate the wavelength ranges that could be used in the near UV range to fragment acidic oligosaccharides, we recorded the first gas-phase optical spectra as a function of laser wavelength for three heparin-derived disaccharides.…”

“…These techniques have been used for the analysis of biomolecules, such as proteins [6 -11], peptides [12][13][14][15][16], and nucleic acids [17][18][19]. In the case of permethylated and fixed-charge derivatized oligosaccharides, the 157 nm UVPD spectra yield intense cross ring fragment ions corresponding to a high-energy dissociation pathway, useful in the assignment of branching and glycosidic linkages [20]. An alternative to UVPD is the reductive amination of oligosaccharides with a fluorophore.…”

The Emerging Field of Diagnostics in Glycosylation Disorders

“…Technological advances in mass spectrometry are promising for improved N-and O-glycan analysis [26]. The ability of Quadrupole Time of Flight (QToF) instrumentation to facilitate MS experiments, especially on glycans which have been derivatised by permethylation, is allowing clear structural assignment of isomeric glycans [27]. Detection of glycans can be improved by using labelling techniques such as the tagging the glycans with fluorophores which increase spectral absorption of glycans, thus improving their detection by high performance anion exchange chromatography -HPLC and ESI-MS methods [28].…”

Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens

“…For a number of years characterization of glycan structures has almost exclusively been the domain of mass spectrometry [19] and in conjunction with nuclear magnetic resonance (NMR) has been used extensively to define novel and unusual glycan structures associated with bacterial proteins [2]. Mass spectrometry as an analytical technique requires that samples to be analyzed are converted into ions in the gaseous phase.…”

“…The glycan moiety generally fragments in one of two ways, either by fragmentation of the bond between two adjacent sugar groups or by cross-ring cleavages occurring via rupture of two bonds within the same sugar unit [19,46]. The nomenclature for fragmentation of carbohydrates is also well established, ions retaining the charge on the nonreducing terminus are classified as A, B and C, those that retain the charge on the reducing terminus are X, Y and Z.…”