Protein Protocols Protein Protocols

812 Hounsell, Davies, and Smith
3. Add 75 µL of methyl iodide (care) and sonicate for 15 min (see Note 2).
4. Extract the permethylated glycans with 1 mL CHCl 3 3 mL H 2O, washing the aqueous
phase with 3 × 1 mL CHCl 3. Wash the combined CHCl 3 washes with 3 × 5 mL H 2O (see
Note 3).
5. Dry the CHCl 3 phase under N 2 after taking an aliqout for liquid secondary ion mass spectrometry
(LSIMS) (see Note 4).
6. Hydrolyze for 1 h in 2 M TFA at 100°C.
7. Reduce samples with 50 mM NaBD 4/50 mM NH 4OH (see Note 5) at 4°C or 4 h at room
temperature.
8. Evaporate once from AcOH and three times from 1:10 AcOH/MeOH.
9. Re N-acetylate samples with 50:50 acetic anhydride/pyridine 100°C, 90 min.
10. Analyze samples by GC-MS on low-bleed 5% capillary column (e.g., HP ultra-2 or
HP5-MS or equivalent), with either on-column or splitless injection and a temperature
gradient from to 50–265°C over 21 min, held for a further 10 min, and a constant gas
flow of 1 mL/min into the MS. Ionization is in EI mode and a mass range of 45–400
(see Note 6).
4. Notes
1. The NaOH can be powdered either in a mortar and pestle or glass homogenizer and thoroughly
vortexed with the DMSO prior to addition to the glycan.
2. If at the end of the methylation a yellow color is present, the reaction can be stopped by
adding a crystal of sodium thiosulfate with aqueous extraction.
3. The permethylated oligosaccharides may also be purified on a Sep-Pak C 18 column
(Waters, Wafford, UK) by elution of permethylated oligosaccharides with acetonitrile or
acetonitrile–water mixture.
4. The reaction with the NaOH/DMSO suspension deprotonates all the free hydroxyl groups
and NH of acetamido groups forming an unstable carbanion. The addition of methyl iodide
then rapidly reacts with the carbanions to form O-Me groups, and thus, permethylate the
oligosaccharide. When subjected to LSIMS, these permethylated oligosaccharides will
fragment about their glycosidic bonds particularly at hexosacetamido residues. This means
each oligosaccharide generates a unique fragmentation pattern allowing the determination
of the oligosaccharide sequence. For example, the oligosaccharide Hex-HexNAc-
HexNAc-Hex will generate the following fragments: Hex-HexNAc and Hex-HexNAc-
HexNAc, where Hex denotes a hexose residue and HexNAc an N-acetylhexosamine.
5. The reduction can also be carried out with 50 mM NaOH, but this is not volatile and is
harder to remove prior to further derivitization.
6. The hydrolysis step generates monosaccharides with the hydroxyl groups involved in the
glycosidic linkages still retaining their protons. Reduction of these monosaccharides with
NaBD 4 will break the ring structure to form monosaccharide alditols with the anomeric
(C 1) carbon being monodeuterated. Acetylation of the free hydroxyls to O-acetyl groups
completes the derivitization. The retention times of the PMAAs on the GC allow the
assignment of the monosaccharide type (galactose, N-acetylgalactosamine, and so on).
On-line mass spectrometric detection identifies fragment ions formed by the cleavage of
C—C bonds of the monosaccharide alditols with the preference: methoxy-methoxy >
methoxy-acetoxy > acetoxy-acetoxy. The resulting spectra are diagnostic for the substitution
pattern, and hence, the previous position of linkage, e.g., a 2-linked hexose will produce
a different set of ions to a 3-linked hexose, and a 2,3-linked hexose being different
again. Selected ions from the spectra of all commonly occurring linkages can be used to
analyze across the chromatogram (selected ion monitoring).