Analysis of Complex Oligosaccharides from Glycopeptides and Glycoproteins Using MSn Spectra and Oligosaccharides Spectral Library

摘要

Glycosylation is a common post-translational modification to cell surface and extra cellular matrix proteins as well as to lipids. Unlike proteins and nucleic acids that are linear polymers of amino acids and nucleotides respectively, with linkages at only one position, carbohydrates can adopt complex branched structures with individual monomeric units linked at one of several sites. A detailed analysis of complex carbohydrate structures has been explored with mass spectrometric techniques, and still presents challenge for the analyst. This presentation will describe a systematic approach of carbohydrates and glycoconjugates analysis with MSn techniques. Oligosaccharides, cleaved from glycoproteins (by hydrazinolysis or enzymatically), were characterized using a hybrid MALDI Ion Trap / TOF mass spectrometer. High mannose, biantennary and triantennary oligosaccharides were analyzed using MS, NS2, MS3 and MS4 modes. Intact oligosaccharides were analyzed in MS mode using a cooling gas to prevent fragmentation. Individual precursor ions were isolated in the trap, subjected to fragmentation with Argon, to provide MS2 data. Product ions were selected for further fragmentation, which was achieved by increasing the energy for collisionally induced dissociation. In MS mode, the singly charged sodium adduct form of these molecules was detected, which is typical of the analysis on conventional MALDI mass spectrometers. In MS2 mode, high mannose oligosaccharides readily lost the core N-acetylglucosamine residues, whilst MS3 and MS4 modes were used to sequentially fragment the product ion corresponding to the residual branched mannose oligomer. In MS2 mode analysis of biantennary and triantennary structures also fragmented losing disaccharide units, such as the galactose - N - acetylglucosamine units that define each antennary branch, or core fucosylated - N - acetylglucosamine units. MS3 of selected MS2 products ions could be used to differentiate fragments generated from either the reducing or non-reducing ends. Cross-ring cleavages were also observed during fragmentation in MS2, and the relevant product ions could be used to differentiate branched structures by further fragmentation in MS3 mode. Many tandem mass spectrometric experiments have been revealing that oligosaccharides might have characteristic signal intensity profiles, depending on the glycosidic linkage and branching structures. In addition to the MSn capability of the platform, there is a library of observational mass spectra acquired from structurally defined oligosaccharides. The presentation will show the enhancement of carbohydrate identification by utilizing comparison procedure of the signal intensity profiles of MSn spectra between the analyte and structurally defined oligosaccharides in the library.