As discussed in Chapter 1, native glycans can be analysed directly by mass spectrometry, but do not ionise as efficiently due to their hydrophilic nature and are thus derivatised prior to analysis by mass spectrometry. Derivatisation methods can either employ a reducing end tag or protect most or all of the functional groups of the carbohydrates.
2.4.8.1 Permethylation
Permethylation involves the exchange of protons in hydroxyl and amide groups for hydrophobic methyl groups and involves the successive base-catalysed ionisation of these functional groups followed by methylation. The technique was introduced by Hakomori and colleagues (Chou and Yang, 1979) who described the use of methylsulphinyl carbanion from dimethyl sulphoxide (DMSO) as the base and methyl iodide as the methyl donor and the chemistry was optimised for high sensitivity application to peptides and carbohydrates by the discovery in isotope dilution MS experiments that the kinetics is on the second timescale rather than the hours and days originally used (Morris, 1972). This led to all subsequent methods utilising permethylation times of minutes in order to minimise byproducts and maximise sensitivity. Permethylation is now commonly catalysed by weaker bases such as sodium hydroxide and has become the staple derivatisation step in many glycomic strategies (Dell, 1990).
For specific oligosaccharide analysis, samples were methylated using the sodium hydroxide procedure. A slurry of NaOH-anhydrous DMSO (1ml) was added to the sample, followed by 0.5ml iodomethane. The reaction mixture was agitated on an automatic multi-tube vortexer for 30mins at room temperature and the reaction quenched by dropwise addition of water. The permethylated glycans were extracted in 1ml of chloroform, washing with 4ml of water (4x) and dried under a gentle stream of nitrogen gas. The resulting glycans were then purified by Sep-Pak® C18 using the aqueous ACN system.
2.4.8.2 Deutero-permethylation
The sample was deuteropermethylated using the sodium hydroxide procedure. A slurry of NaOH-anhydrous DMSO (1ml) was added to the sample, followed by 0.5ml deuterated iodomethane-d3. The reaction mixture was agitated on an automatic multi-tube vortexer for 30mins at room temperature and quenched by dropwise addition of water. The deuteropermethylated glycans were extracted in 1ml of chloroform, washing with 4ml of water (4x) and dried under a gentle stream of nitrogen gas. The resulting glycans were then purified by Sep-Pak® C18 using the aqueous ACN system as described below.
2.4.8.3 Reduction
To reduce the oligosaccharides prior to methylation, they were incubated with 10mg/ml of NaBH4 (in 2M aqueous NH3) at room temperature for 2hrs. After borate removal with acidified methanol co-evaporation, they were permethylated as described previously (Section 2.4.8.1).
2.4.8.4 Deutero-reduction
To deuteroreduce the oligosaccharides prior to methylation, they were incubated with 10mg/ml of NaBD4 (in 2M aqueous NH3) at room temperature for 2hrs. After borate removal with acidified methanol co-evaporation, they were permethylated as described previously (Section 2.4.8.1).
2.4.8.5 Sep-Pak® clean up: Aqueous acetonitrile system
Purification of the permethylated sample was achieved by reverse-phase chromatography using C18 Sep- Pak® cartridges. Sep-Pak® Classic C18 cartridges were attached to 10ml glass syringes and conditioned successively with 5ml of methanol, water, ACN and 15ml of water. The sample was dissolved in 200µl of 50% (v/v) H2O/MeOH and loaded onto the cartridge. The sample was washed with 5ml of water and then eluted stepwise with 3ml of each 15%, 35%, 50%, 75% and 100% (v/v) aqueous ACN. The fraction volumes were reduced on the SpeedVac® and lyophilised.
2.4.8.6 TMS derivatisation
For sugar composition, trimethylsiylated (TMS) derivatives of the polysaccharides were prepared by methanolysis (1M methanolic-acetylchloride, 80ºC, 16hrs) and dried under nitrogen. The samples were re-N- acetylated (methanol/pyridine/acetic acid (50/1/5, v/v/v), room temperature, 15mins) with the reagent removed by drying under nitrogen. Trimethylsiylation was carried out with tri-Sil ‘Z’ Derivatising agent (room temperature, 30mins). The samples were purified by centrifugation and the supernatant was re-dissolved in hexanes prior to GC-MS analysis.
2.4.8.7 Alditol acetates
For compositional analysis by alditol acetate, the non-permethylated samples were hydrolysed (2M TFA, 121ºC, 2hrs), dried under nitrogen and the monosaccharides reduced (10mg/ml NaBD4 in 2M aqueous NH3, room temperature, 2hrs). After borate removal with acidified methanol co-evaporation, they were re- acetylated (acetic anhydride, 100ºC, 1hr), purified by washing with chloroform and water (4x) and prepared for GC-MS by suspension in hexanes.
2.4.8.8 Linkage analysis
Linkage information was achieved by the analysis of partially methylated partially acetylated alditol acetate derivatives. Samples were permethylated as described followed by hydrolysis (2M TFA, 121ºC, 2hrs) and reduction (10mg/ml NaBD4 in 2M aqueous NH3, room temperature, 2hrs). After borate removal with acidified methanol co-evaporation, the monosaccharide samples were re-acetylated (acetic anhydride, 100ºC, 1hr), purified by washing with chloroform and water (4x) and prepared for GC-MS by suspension in hexanes (Albersheim, 1967).
2.4.8.9 Fatty acid methyl ester analysis
Fatty acid methyl esters were produced for lipid analysis and samples were hydrolysed in 200μl methanolic- acetylchloride (1M) overnight in 80ºC and the reaction terminated by drying under a stream of nitrogen. The samples were dissolved in chloroform and washed several times with water (4x), then dried under nitrogen and ultimately prepared for GC-MS by suspension in hexanes.
2.4.8.10 Esterification of carboxylic groups using methanolic HCl
This procedure was employed for the identification of the 190Da moiety contained in the N-glycan of the H. volcanii S-layer glycoprotein. The digested glycopeptides were incubated with 2M HCl/MeOH for 15mins, dried under nitrogen, then purified by nanoLC and analysed by MALDI-TOF.