As an immunologist and analytical scientist, I have always been fascinated with the immune system and how pathogens and the immune system have evolved hand-in-hand as the former seeks new ways to avoid and disguise itself from the latter and the immune system develops alternative mechanisms to seek out and destroy invading pathogens. It’s a miniature arms race occurring inside each and every one of us, but only really becoming noticeable to us when we feel the effects of the raging battle through illness. I do believe that the immune system represents a model and spell-binding system in which to view evolution occurring, although my young children would probably disagree and much prefer an alternative bedtime story to one of my favorite books for children, titled, Your Amazing Immune System! (details of how to get a free copy of this book if you wish to go with the immunological children’s bedtime story.)
Glycosylation plays an enormous part in the correct functioning of the immune system as well as in enabling pathogens to evade the immune system and again you can see evolution at work here as both the immune system and pathogens adopt different and ever more complex glycosylation patterns to try and gain an advantage over each other. Glycosylation is of vital importance to the immune system as it plays a crucial role in a number of processes such as antibody function, both T- and B-cell receptor signaling, pathogen recognition, immune cell trafficking and my favorite, apoptosis. On the opposite side of the coin, pathogens use glycosylation as a mechanism of disguise to shield themselves from the immune system weaponry, as a tool to attach and enter cells and even by altering the hosts glycosylation machinery to allow its escape from the immune system. The analysis of glcosylation thus represents an interesting research area as a greater understanding of the glycan structure and how this relates to specific functions could lead to targeted interventions that could tip the balance of favor in the direction of the immune system over the pathogen.
Glycan analysis can be performed using a variety of analytical chemistry techniques, however the most commonly employed method is HPLC coupled to mass spectrometry of enzymatically or chemically released glycans. A number of different chromatographic separation and detection methods can be employed for glycan analysis, for example:
- High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD): You might find Technical Note 20, Analysis of Carbohydrates by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAE-PAD), (downloadable PDF), of interest on this topic.
- This recent webinar, now available on-demand, titled, Oligosaccharide Analysis Using High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (link to short registration page after which webinar begins to play) gives a great overview of this approach.
- The Hydrophilic Interaction Chromatography (HILIC) technique can be used with a fluorescence detector for purified glycans derivatised at their reducing end with a fluorochrome.
- New mixed-mode HPLC columns for the separation of both neutral and charged glycans as I discussed in this recent post, titled, Isomeric Separation & Structural Identification of Labeled N-linked Glycans.
Each separation method has its own advantages and should be matched with the questions being sought and the type of analysis required. However, irrespective of the method used, HPLC is a powerful tool for glycan analysis which is helping to unravel the glycosylation pattern of the immune system and associated pathogens, moving us closer to interventions that help the immune system win the battle.
Like what you are learning?
What are your views on immune system glycosylation? Do we currently have the right tools at our disposal to efficiently perform glycan analysis? Are you using HPLC for glycan analysis? I’d like to hear you experiences and comments.
Timothy Cross is a regional marketing manager within EMEA for HPLC in the Chromatography and Mass Spectrometry Division at Thermo Fisher Scientific Inc. As a former scientist and following his Professional Diploma and Postgraduate Professional Diploma in Marketing from the Chartered Institute of Marketing (UK), Timothy has worked in a variety of roles in the life science industry including R&D scientist, product manager, marketing manager and demand generation manager. This background enables Timothy to pursue his passion of truly understanding the scientist’s application needs and requirements and delivering the appropriate workflows, support and total solutions to drive and enhance these applications. Timothy received his B.Sc. in Biological Sciences and M.Sc. and Ph.D. in Immunology from the University of Birmingham (UK).