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

2. 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

3. 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.

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

5. 2-AA vs. 2-AB Reaction of 2-AA and 2-AB with glycans 2-AA 2-AB

6. 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 1214161820222426283032343638404244 Mono Di 0 2 4 6 8 10 12 AAAB Transferrin

7. 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

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

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

10. Effect of Time on Sensitivity

11. 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

12. Effect of different buffers on sensitivity for 2-AA

13. 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

14. 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.

15. 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 83 62 47.5 32.5 G Control +PNGase -TX114+TX114

16. 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).

17. 10111213141516171819202122232425262728 CHPV control CHPV + NBDNJ CHPV 24hrs prior CHPV +NBDNJ 24hrs prior Minutes HPLC Analysis of CHPV G-protein Glycans G3M7N2

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

19. 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)

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