glycosylation analysis by UHPLCMost of my early research career focused on the post-translational modifications (PTMs) of proteins and in those days there was only one worthy PTM in my eyes and that was phosphorylation while glycosylation hardly registered. That was because phosphorylation governed important cellular processes (in my view at least) such as cell signalling; however, my love of phosphorylation slowly waned as I grew to understand the only reliable method to measure phosphorylation was with the potentially harmful radioactive Phosphorus-32 (32P)! (Link to Wikipedia page.) That’s when I started to get more interested in glycosylation and the huge diversity of modifications this could entail, after all phosphorylation was only adding phosphate on to a serine, threonine or tyrosine. Glycosylation offered more possibilities and intrigue and the opportunity to use other analytical techniques that didn’t involve 32P.

Glycosylation is one of the most widely studied and common post translational modifications of eukaryotic proteins, with estimates ranging from 20% to over 50% of proteins being glycosylated. Post translational modifications lead to the huge proteomic diversity observed and are critical to the correct functioning of many cellular and systems processes such as cell signalling, immune function and cell replication/proliferation as I discussed in my previous blog post, titled, Glycan Analysis with a Particular Focus on the Immune System, (link to post). With glycans, the structure of the oligosaccharide is a critical element in how the glycan functions and its impact on cellular processes. Hence understanding amounts and composition of glycans is of critical importance in determining the precise function of glycoproteins, but also when developing glycoprotein-based therapeutics. A number of analytical techniques have been employed in glycan analysis to determine this sort of information and are summarized under these four categories here. And, what’s more, you don’t need 32P!

  • Monosaccharide Analysis – This analysis is typically performed to determine monosaccharide composition as well as to quantify the amounts of individual monosaccharides. Neutral monosaccharides can be partially or weakly ionized under basic conditions and separated by anion-exchange chromatography columns (Thermo Scientific Dionex CarboPac columns) combined with electrochemical detection, in a technique known as HPAE-PAD (link to downloadable technical note discussing the technique). For charged monosaccharides, HPAE-PAD can also be used or the released monosacchardes can be derivatized and analyzed using HPLC with fluorescent detection.
  • Glycan Analysis – Information on glycan patterns and structure is particularly valuable and structural elucidation is done after the enzymatic or chemical release of glycans from the protein. Again, HPAE-PAD can be used for such glycan analysis or HPLC with fluorescent detection, typically using HILIC column chemistries. More recently mass spectrometry has emerged as a powerful tool for the structural elucidation and quantitation of glycans.
  • Intact Glycoprotein Profiling – This can be employed to determine the expected pattern and degree of glycosylation. Its significance has grown in recent years with the interest in recombinant monoclonal antibodies as biotherapeutics where the glycosylation of the antibody has to be fully characterized to ensure safety and efficacy. Intact glycoprotein profiling requires a high resolution, accurate mass, mass spectrometer(Thermo Scientific Orbitrap-based mass spectrometers). Intact glycoprotein profiling can be performed with direct infusion, or with very complex samples liquid chromatography separation is typically required prior to mass spectrometry.

These techniques are aiding our understanding of the complex glycoproteome and allowing us to obtain more accurate and comprehensive data with improved efficiency. Improving our knowledge of glycosylation and other post translational modifications will advance our understanding of health and disease and the development of potent biopharmaceuticals.

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By the way, the image is of protein p53 binding to a strand of DNA; in large numbers of cancers, this protein is rendered inactive by mutations.

Additional Resources

  • Do check out our comprehensive glycomics resources and information on our comprehensive Glycomics Community Page featuring numerous on-demand webinars, downloadable applications and technical notes, and more.
  • Download a complete guide to glycan analysis.
  • Request a complimentary copy of a glycan analysis reference card which provides a handy reference of common monosaccharides and subsituents to assist you in quickly interpreting your spectra and determine the value of a glycan composition.

What do you envisage will be the next major development in glycan analysis? Are the current analytical techniques sufficient for glycan analysis – where could they be improved? Is there sufficient focus and funding for post-translational modification research in your opinion? I would like to hear your views.

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).