Chip electrospray mass spectrometry for carbohydrate analysis

Currently two types of chip systems are used in conjunction with MS: out-of-plane devices, where hundreds of nozzles, nanospray emitters are integrated onto a single silicon substrate from which electrospray is established perpendicular to the substrate, and planar microchips, embedding a microchannel at the end of which electrospray is generated in-plane, on the edge of the microchip. In the last two years, carbohydrate research greatly benefited from the introduction and implementation of the chip-based MS. In two laboratories the advantages of the chip electrospray in terms of ionization efficiency, sensitivity, reproducibility, quality of data in combination with high mass accuracy, and resolution of detection were systematically explored for several carbohydrate classes: O- and N-glycopeptides, oligosaccharides, gangliosides and glycoprotein-derived O- and N-glycans, and glycopeptides. The current state-of-the-art in interfacing the chip electrospray devices to high-performance MS for carbohydrate analysis, and the particular requirements for method optimization in both positive and negative ion modes are reviewed here. The recent applications of these miniaturized devices and their general potential for glycomic-based surveys are highlighted

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

A thin chip polymer-based microsprayer has been coupled to a hybrid quadrupole time-of-flight mass spectrometer (QTOF MS) and introduced in carbohydrate research. The feasibility of the approach is demonstrated for mapping, sequencing and structural elucidation of glycoconjugates originating from human body fluids and tissues such as a glycopeptide mixture from normal human urine and an isolated and purified GT1 ganglioside fraction from normal adult human brain. The optimization procedure required by each glycoconjugate category is described and the advantages of the system in terms of flexibility and adaptability to QTOF MS, stability of the ESI MS signal, carbohydrate ionization and sequencing, sensitivity, speed of analysis and sample consumption are discussed

Application of the 50% Hydrazine Solution Method for O-Glycans Release, their Chemical Labeling, and HPLC Separation

Mucins are high molecular mass glycoproteins with oligosaccharides O-bonded to the protein core. β-elimination is the most popular method used for releasing of O-glycans. However to such released glycoforms it is difficult to introduce a label to amplify a signal for oligosaccharide detection

STRUCTURE DETERMINATION OF N- AND O-GLYCANS USING SOFT IONIZATION MASS SPECTROMETRY

Glycosylation is a crucial event in the post-translational modification of proteins. Numerous investigations have shown clearly that both the primary structure and the conformation of glycoproteins are intimately connected with their biological function and metabolic fate. Many laboratories are currently directing their efforts towards the development of more specific and sensitive methods for the determination of the primary and secondary structure of glycopeptides as a first step, towards the analysis of their biological function. In view of the complexity of the structures involved, it is evident that only a combination of different methods will yield the desired results. In this instance soft ionization mass spectrometry and high resolution NMR have proved to be expecially useful in determining such different structural parameters as, for example, the type and number of sugar components, the sequence of sugar residues, the sites of the glycosidic linkages and their anomeric confuguration, the sites of glycosylation on the peptide chain, and the 3D-conformation of these structures. Glycans linked N-glycosidically through a chitobiosyl-Asn linkage are usually released enzymatically from the peptide chain prior to analysis ‚whereas O-glycans, e.g., of the mucin type with a GalNAc (al-3)-Ser/Thr linkage, can be released by alkaline borohydride treatment. In this contribution special emphasis will be given to the potential of mass spectrometric methods in analysing the complex mixtures that are obtained after enzymatic release of N-glycans from a single glycosylation site, or after alkaline borohydride treatment of mucin O-glycans