Determining the Structure of Glycan Moieties by Mass Spectrometry

Mass spectrometry provides a complementary method to HPLC (unit 12.6) for the analysis of glycans. In general, its resolution, defined as its ability to detect carbohydrates in a complex mixture, is higher than in HPLC, particularly for larger structures. In addition, it provides a mass from which the composition of the glycan, in terms of its isobaric monosaccharide composition, may usually be determined directly. Analysis by mass spectrometry is also faster than by HPLC. On the other hand, mass spectrometry does not usually allow isomeric compounds to be distinguished unless they give diagnostic fragmentation spectra, and, because mass spectrometry is more susceptible to the presence of salts and residual buffers, efficient sample preparation is much more critical and sensitivity generally lower. The high sensitivity and specificity of HPLC is a result of the fluorescent derivatives that are necessary for detection. Although the same technique of derivative preparation can be applied to mass spectrometry in order to improve detection, there does not yet appear to be a single derivative that improves detection in both techniques. Because of the need to analyze minute amounts of sample, there is not usually enough for the preparation of two sets of derivatives, and the first choice for sample analysis is usually the more sensitive technique of HPLC coupled with exoglycosidase digestion.

Intact glycans may be detected directly as singly charged ions by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS; see Basic Protocol 1 and unit 16.1; Harvey, 1999) or, following permethylation or peracetylation, by fast atom bombardment mass spectrometry (FAB-MS; see Basic Protocol 2). Electrospray ionization mass spectrometry (ESI-MS; see Basic Protocol 3) also gives good signals for the smaller glycans, but produces singly, doubly, or even triply charged ions, depending on the conditions. This technique is not as good as the other two for glycan profiling; additionally, the sensitivity tends to fall as the glycans become larger. All three techniques give ions in the form of adducts, usually with sodium adducts in the case of MALDI-MS, or as mixtures of sodium and hydrogen in the case of ESI-MS. Adducts can be controlled by addition of organic acids (to promote protonation) or suitable salts to the MALDI or FAB matrices or to the electrospray solvent. For rapid profiling, MALDI-MS is the most appropriate technique. For obtaining structure-revealing fragment ions, FAB-MS or ESI-MS followed by collisional activation gives superior results.

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Good Carb Diet

Good Carb Diet

WHAT IT IS A three-phase plan that has been likened to the low-carbohydrate Atkins program because during the first two weeks, South Beach eliminates most carbs, including bread, pasta, potatoes, fruit and most dairy products. In PHASE 2, healthy carbs, including most fruits, whole grains and dairy products are gradually reintroduced, but processed carbs such as bagels, cookies, cornflakes, regular pasta and rice cakes remain on the list of foods to avoid or eat rarely.

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