Sample glycans

Some of the multibranched complex glycans of hen ovomucoid have been analyzed by f.a.b.-m.s. together with e.i.-m.s. These glycans exhibit exceptional structural features, because their mannotriose core can be highly substituted, as shown in 25. All samples gave abundant [M+Na]" ions when analyzed as their permethylated alditols, and this permitted the... 

Purification of the conjugates may be done by reverse phase HPLC separation. Dry the reaction solution under a nitrogen stream and reconstitute in a minimum volume of acetonitrile/water (1 1, v/v). Apply the sample to a 5 pm Cig-silica HPLC column (250 X 4.6 mm, Nucleosil). Elute with a gradient of water to acetonitrile at a flow rate of 1 ml/minute over a time course of 30 minutes. Free BNAH and BNAH-glycan derivatives can be monitored by absorbance at 275 nm. The conjugate peak also will be positive for carbohydrate by reaction with orcinol, which can be detected by spray after spotting a small eluted sample on a TLC plate. 

Methylation analysis (and its different applications) is one of the most widely used methods in the structural characterization of carbohydrate chains (reviewed in refs, 5-8). In the present article, the progress achieved, and the problems encountered, in the analysis of animal glycolipids and glycoproteins are discussed, although many of the topics also concern the analysis of carbohydrate chains in general. Emphasis is laid on the efforts made to find ways to lessen the amounts of sample needed for the analysis, because only small amounts of glycan samples isolated from biological sources are often available. 

The partially methylated monosaccharides obtained on depolymerization of the permethylated sample are preferably analyzed as acetates by g.l.c.-m.s., as shown by Bjomdal and coworkers.41,42 The neutral sugars and the amino sugars obtained in acetolysis-acid hydrolysis are reduced, and acetylated for the analysis, and the amino-hexitol and the neuraminic acid residues are acetylated after methanolysis. Identification with the aid of g.l.c.-m.s. has been described for all of the common components of protein- and lipid-linked glycans and oligosaccharides from animal cells, namely, the neutral sugars,41-43 hexitols,44 hexosamines,29,43,45,46 aminohexitols,31,32 and neuraminic acids.33,34,47... 

Zhuang et al. [152] derivatized glycan samples with 8-aminopyrene-l,3,6-trisulfonic acid to get a charge for electrophoresis and a fluorescent label for detection. They analyzed glycan derivatives in the blood of cancer patients... 

Fig. 3. Schematic of staining process of SARS-CoV immunochip. (1) Spotting A high-precision robot transfers the samples, SARS-CoV proteins, and glycans of various complexities, from 96-well plate to nitrocellulose-coated glass slides. (2) Staining Before staining, the slides are rinsed with IX phosphate-buffered saline (PBS), and blocked with 1% bovine serum albumin (BSA)-PBS containing 0.05% NaN3 and 0.05% Tween-20. They are subsequently incubated with horse anti-SARS sera. The primary antibodies captured by microarrays are detected using biotinated anti-horse immunoglobulin (Ig)G, and visualized by Cy3-streptavidin.

---