The development of a robust method for labelling glycans & its practical application. Glycobiology Institute University of Oxford

Current methods of analysis Mass Spectroscopy TLC NMR HPLC analysis: –Released glycans from glycoproteins can be separated into component species using HPLC. Normal phase, Ion exchange & reversed phase commonly used. –These can be used with fluorescently- labelled oligosaccharides. Enzyme Digestion

Objectives To produce an improved labelling method – higher sensitivity – ease of use – high-throughput To check the viability of this method in a biological context.

Factors to consider 2-AA vs. 2-AB Volume Time Temperature Presence of water Labelling buffer Post-label purification

2-AA vs. 2-AB Reaction of 2-AA and 2-AB with glycans 2-AA 2-AB

2-AA vs. 2-AB Published OGS conditions –30% DMSO, 70% Acetic Acid, 0.35M label & 1M Sodium cyanoborohydride (5  l) –65 o C for 2hrs –Clean up using Glycoscience S cleanup cartridge Minutes Mono Di AAAB Transferrin

Should water be used? Literature suggests that 10% water may be used. Range of water concentrations were tested. Samples, post-labelling, purified using DPA-6S SPE columns (greater recovery than cellulose). 20% Water appears optimal

What reaction volume should be used? Reaction volumes were varied to see whether this has an effect on sensitivity.

Temperature and Time Is the temperature used optimal? – Two temperatures 65 and 80 o C tested (commonly used in lab)

Effect of Time on Sensitivity

Effect of Buffers It is suggested that 2-AA labelling can be performed in several buffers –OGS conditions – 4% sodium acetate, 2% boric acid in methanol – acetic acid (1-10%) in methanol or acetonitrile – water

Effect of different buffers on sensitivity for 2-AA

Preliminary Conclusions For practical use, the method used should be: –50  l volume –20% water –65 o C for 2 hours –DMSO/Acetic acid –May be performed directly on protein- containing buffered solutions, e.g. PNGase F digests

Viral G-protein glycan characterisation Chandipura virus (CHPV) is a member of the genus Vesiculovirus –It is Athropod-transmitted and can infect humans –CHPV infection is widespread in both humans & animals throughout India. –Interesting biological example of viral G-protein. –G protein of CHPV is found to be 1706 nucleotides in length with molecular weight 66kD.

Viral G-protein glycan characterisation Virus was added to MDBK cells for 24 hrs Hi-speed spin (200,000g) to pellet viral particles. TX114 separation & protein precipitation to ensure membrane protein only present. PNGase added to release N-linked glycans. M G Control +PNGase -TX114+TX114

Effect of NB-DNJ Does NB-DNJ have an effect on viral glycan synthesis in ER? –Cells were infected with CHPV. –Some cells were incubated with NB-DNJ 24hrs prior to infection. –Others were co-infected with NB-DNJ. –Control (non-NB-DNJ).

CHPV control CHPV + NBDNJ CHPV 24hrs prior CHPV +NBDNJ 24hrs prior Minutes HPLC Analysis of CHPV G-protein Glycans G3M7N2

Triplicates Transferrin (FOS)  1 acid -glycoprotein* Fetuin*  1 Anti-Trypsin*

Conclusions The DMSO/Acetic acid method has been improved, as revealed by analysis of N-linked glycans, dextran. 2-AA is more sensitive than 2-AB The addition of water ensures improved labelling in the DMSO/Acetic acid protocol where the volume of reagants should be 50  l with 20% water present. The reaction should progress at 65 o C for 2-3 hrs The reaction can be performed directly on PNGase F digests of glycoproteins - no post digest cleanup required Post-labelling purifcation performed by SPE (amenable to 96 well plate procedure)

Acknowledgements Dr Terry Butters Dr David Neville Dom Alonzi With thanks to Professor Raymond Dwek