Carbohydrate Analysis With Gas Chromatography-Mass Spectrometry

Carbohydrates are energy-rich molecules which provide energy for life

processes and the building parts of the cellular structure of plants and animals
(Lindhorst, 2003). They play crucial roles in the immune system, pathogenesis,
blood clotting, fertilization, and protein folding and placement. Apart from this,
carbohydrates get added to proteins as a result of post translational
modification called glycosylation. Now we know that virtually all extracellular
proteins and some cytoplasmic proteins get glycosylated (Oxley et al. 2004).
Many analytical techniques have been developed to study the structure of
carbohydrates but due to their complexity not a single technique can offer
comprehensive information. In most cases the target molecules are modified
prior to analysis. The incredible diversity of these carbohydrate molecules can
be explained in the following example. Two identical hexoses can generate 20
isomeric glycans and three identical hexoses can form 448 isomers. On
comparing this complexity to that of amino acids in proteins, only a single
peptide can be formed from three identical amino acid residues (Oxley et al.
2004). Here we will be using GC-MS (Gas Chromatography coupled with Mass
Spectrometry) to analyze monosaccharides in the total cell wall extracts of
spinach leaves. The extracts will be derivatized by trimethylsilane (TMS) before
analysis on the GC-MS instrument.
Lindhorst TK. 2003. Essentials of Carbohydrate Chemistry and Biochemistry.
Wiley-VCH: Weinheim.
Oxley D, Currie G C, Bacic A (2004) Analysis of carbohydrate from
glycoproteins. In: Purifying proteins for proteomics: Purifying proteins for
proteomics: A laboratory manual. R J Simpson (ed), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NewYork, USA, ch 25, pp 579-636.

Cell wall preparation by the Alcohol Insoluble Residue (AIR) method

10 g of Spinach leaves were weighed.
Tissue was pulverized in liquid nitrogen to obtain a fine powder.
Then it is boiled for 30 min in 96% ethanol.
Then it is centrifuged at 10,000g for 5 min.
1

The supernatant was removed and the pellet was retained.

The pellet was washed in 70% ethanol and subsequently centrifuged (10,000g
at 5mins) until it appears colorless and free from chlorophyll.
A final wash with 100% acetone was performed, and the pellet was air dried.

The protocol is adapted from Jesper Harholt, Jacob Krger

Jensen,Susanne Oxenbll Srensen, Caroline Orfila, Markus Pauly, and
Henrik Vibe Scheller (2006) ARABINAN DEFICIENT 1 Is a Putative
Arabinosyltransferase Involved in Biosynthesis of Pectic Arabinan in
Arabidopsis. Plant Physiol. 140: 49-58.

Hydrolysis of glycans from Glycoproteins

1 Add 80 l of 1 M methanolic HCl followed by 40 l of methyl acetate to each
tube.
2. Seal each tube with a cap, liner, and Teflon tape.
3. Vortex each mixture thoroughly. Heat the samples and standards for 16
hours at 80C.
4. Remove the tubes from the heat and cool them to room temperature before
opening the vials.
5. Add 500 l of t-butyl alcohol to each tube and vortex them thoroughly.
Immediately evaporate the solvent by passing a stream of argon (or nitrogen)
over the solution at room temperature.
6. N-acetylate the samples by adding 50 l of super-dry methanol, 5 l of
pyridine, and 5 l of acetic anhydride and mix thoroughly.
7. Incubate the tubes for 15 minutes at room temperature.
8. Evaporate the samples to dryness by passing a stream of argon (or nitrogen)
over the solution at room temperature.
9. To a thoroughly dry sample, add 100 l of Tri-Sil. Incubate the tube for 10
minutes at 70C.

10. Resuspend the samples in 1ml of DCM (Dichloromethane) and add it to

GC-MS sample vials.
This protocol is modified from the protocol in Oxleys paper.
Oxley D, Currie G C, Bacic A (2004) Analysis of carbohydrate from
glycoproteins. In: Purifying proteins for proteomics:, Purifying proteins for
proteomics: A laboratory manual. R J Simpson (ed), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NewYork, USA, ch 25, pp 579-636.