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

Isolation and structural characterization of sialic acid-containing glycopeptides of the O-GlycosidicType from the urine of two patients with an hereditary deliciency in α-N-acetylgalactosaminidase activity

Glycopeptides have been isolated fromthe urine of two patients, aged 5 and 6, with a newlysosomal storage disease characterized by a defi-ciency in α-Ν-acetylgalactosaminidase activity. Isola-tion of these glycopeptides was achieved using gel fil-tration and ion-exchange chromatography. Structuraldetermination was done using one- and two-dimen-sional 500 MHz 1H-NMR spectroscopy and FAB mass spectrometry of native and derivatized glyco-peptides. The following structures were inferred asbeing present: Glycopeptide A (up to 140 mg/l urine)(l)-(3)Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GalNAcα1l- RAl:R = Ser A2:R=Thr A3:R=Thr-ProGlycopeptide Β (up to 80 mg/l urine)(4)-(6)Neu5Acα2-3Galβ1-4GlcNAcβ1-6(Neu5Acα2-3--Galβ1-3)GalNAcα1-R Bl:R = Ser B2:R=Thr B3: R =Thr-Pr

Structures of neutral O-linked polylactosaminoglycans on human skim milk mucins. A novel type of linearly extended poly-N-acetyllactosamine backbones with Gal beta(1-4)GlcNAc beta(1-6) repeating units

O-Linked oligosaccharides were isolated from human skim milk mucins and from mucin-derived glycopeptides by reductive beta-elimination. The released alditols were fractionated by DEAE-Sephadex chromatography and purified by high performance liquid chromatography on primary amine bonded phase. The structures of the major neutral oligosaccharide alditols could be established by fast atom bombardment and electron impact mass spectrometry, combined with methylation analysis, 500-MHz 1H nuclear magnetic resonance spectroscopy, and endo-beta-galactosidase (from Bacteroides fragilis, EC 3.2.1.103) digestion (where n = 0-3): (formula; see text) Major O-glycosidically linked oligosaccharides on skim milk mucins are of the Gal beta(1-3)[GlcNAc beta(1-6)] GalNAc core type 2 and exhibit linearly extended backbone chains of the poly N-acetyllactosamine type comprizing up to at least four repeating units, which are linked by the hitherto unknown sequence GlcNAc-beta(1-6) Gal rather than GlcNAc beta(1-3)Gal. A considerable portion of neutral alditols is represented by branched isomers of the linear species, which are distinguished by their content of 3,6-disubstituted galactose and their partial resistance to endo-beta-galactosidase digestion

Purification and structures of branched blood‐group‐B‐active glycosphingolipids from human erythrocyte membranes

Three different variants of complex, branched, highly blood-group-B-active glycosphingolipids (B-III, B-IV, and B-V) have been isolated from human erythrocytes by means of partition of their membranes in n-butanol/phosphate buffer, subsequent removal of nonpolar lipids and proteins by several steps of phase distribution, acetone or sodium acetate precipitation, peracetylation and repeated fractionation of all crude extracts by silicic acid and ion exchange column chromatography. Finally, peracetylated B-glycolipid fractions were purified to homogeneity by preparative silica gel high-performance thin-layer chromatography. Their structures were elucidated by gas chromatographical sugar analysis, by combined gas chromatography/mass spectrometry of partially methylated alditol acetates for the identification of glycosidic linkages, and by fast atom bombardment and electron impact mass spectrometry of the undegraded, permethylated substances in order to establish the molecular mass, sugar sequence, type of oligosaccharide chain, position of hexosyl branching points, number of N-acetyllatosamine units, as well as sphingosine and fatty acid patterns of the ceramide residues. 360-MHz 1H nuclear magnetic resonance spectroscopy in (2H)dimethylsulfoxide of deuterium-exchanged native B-III and B-IV identified all carbohydrate components, their sites of attachment, the anomeric nature of their glycosidic linkages and the sequential arrangement within the oligosaccharide chain. Furthermore, it established the nature of branching points within the carbohydrate sequence, and assigned the different typical saccharide branches to either the position 2 versus 3, or position 3 versus 6 of the 2,3-disubstituted or 3,6-disubstituted galactoses. The nature of the anomeric linkages and branching points of B-V was based upon the series of NMR data obtained from the B-I--B-IV analogues. All results thus establish the following structures