Detection-Fragments-Masses-Presence-Number-Deficiency-Enzymes-Degradation-Moieties-Patient-i

lacto-n-neotetraose describes the derivatization of oligosaccharides present in urine with phenyl-1-methylpyrazolone, which renders them hydrophobic, thus allowing desalting with Combi cleanup columns prior to injection. This method allows the detection of storage of oligosaccharides, which may indicate the presence of one of the infantile Pompe disease, α-mannosidosis, Gm1-gangliosidosis, Sandhoff disease, sialidosis, galactosialidosis, I-cell disease, and Hydrazinonicotinic acid as a novel matrix for highly sensitive and selective MALDI-MS analysis of oligosaccharides.of Public Health, Fudan University, Shanghai 32, P. R. China. Analysis of oligosaccharides with matrix-assisted laser desorptionionization mass spectrometry (MALDI-MS) remains challenging due to their low ionization efficiency.

The sensitivity achieved by MS for oligosaccharides lags far behind that for proteinspeptides. Here, hydrazinonicotinic acid (HYNIC) is proposed as a new matrix to realize highly sensitive and selective analysis of oligosaccharides in MALDI-MS. The detection limit of maltoheptaose provided by HYNIC is as low as 1 amol, which is five orders of magnitude lower than that provided by the traditional matrix 2,5-dihydroxybenzoic acid (DHB). Interestingly, HYNIC displayed remarkable selectivity for ionization of oligosaccharides, making glycans from glycoproteins become more accessible to be detected even without pre-purification, as demonstrated by the direct detection of the oligosaccharides from human serum without pre-separation of the proteinspeptides. The HYNIC matrix also possessed the virtue of higher homogeneity of crystallization and better salt tolerance (up to 0 mM NaCl, 1 mM urea and mM sulfocarbamide et al.) compared with the traditional matrix DHB. Furthermore, the HYNIC matrix afforded adequate fragmentation, thus providing rich information for the structural elucidation of the oligosaccharide.

Therefore, using HYNIC as the matrix to directly analyze oligosaccharides is inherently simple and straightforward.Comparison of separation modes of high-performance liquid chromatography for the analysis of glycoprotein- and proteoglycan-derived oligosaccharides. J Chromatogr A 1996 Aug 9;741(1)147-8.Porous graphitised carbon (PGC) has been explored for high-performance liquid chromatography (HPLC) of mono- and di-saccharides released from proteoglycans and of fluorescently labelled oligosaccharide derivatives for high-sensitivity detection. Sulphated oligosaccharides show good retention and separation behaviour on PGC-HPLC, and compared to anion-exchange or reversed-phase ion-pair chromatography the chromatography is carried out in the absence of salt. Due to their poor retention on PGC-HPLC the analysis of single uronic acids has been optimised with high pH anion-exchange chromatography. Fluorescent labelled derivatives formed by reductive amination of neutral oligosaccharides with 2-aminobenzamide have been chromatographed on PGC-HPLC and by BioGel P4 gel Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.

Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe lacto-n-neotetraose of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D4A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp4. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D4A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc.