Glycans are a hot topic in research because they are so common in biological systems, yet complex and notoriously problematic to analyze. Two pieces of glycan news crossed my desk this week, so I had to blog about them.
First, which I talk about here, is the introduction of a new column for glycan analysis (Thermo Scientific GlycanPac AXH-1 column) by HPLC, UHPLC, or LC-MS that promises to be a game-changer in glycan analysis. In Part II of this blog, I will talk about a great glycan webinar I attended called Glycan Analysis, An Expert Overview of Glycomics Techniques & Workflow Strategies that delivers just what the title says—an overview of techniques for glycan analysis.
First, it seems important to reflect on why glycans are important to analyze. In biological systems, glycans are found widely in free states as well as glycosolated to proteins and lipids. I was surprised to discover an estimated 50% of all proteins are glycosylated, and they are the most prevalent type of post-translational modification—even more than phosphorylation, methylation, or acetylation. Glycans are involved in all sorts of biological processes—recognition and regulatory functions, cellular communication, gene expression, cellular immunity, growth and development, and pathogen interaction to name a few. It therefore is no surprise that aberrant glycosylation is implicated in a multitude of diseases, from schizophrenia to cancer and metastasis. Glycans are important in therapeutic protein drug development as well since there is strong evidence that bioactivity and efficacy are affected by glycosylation.
Although studying glycosylation is important for basic, disease, and therapeutic research, the complexity of carbohydrates makes analysis is problematic. For HPLC analysis, glycan methods range from normal phase (NP) and hydrophilic interaction (HILIC) chromatography, to ion-exchange (IEX) chromatography and reversed-phase (RP) chromatography. If you’ve ever done glycan analysis by HPLC, you have probably found coeluting peaks because of the wide mix of glycan attributes.
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What’s different about the new glycan column is that it uses a combination of modes—charge, size, and polarity—to separate glycans. It uses a mix of both weak anion-exchange (WAX) and HILIC retention mechanisms—while WAX selectively retains negatively charged glycans, HILIC separates similarly charged glycans according to their polarity and size. This column is a big step forward in resolving those components. The column’s data sheet (downloadable PDF) shows example separations using different LC-fluorescence and LC-MS methods. Separation by HPLC and UHPLC uses 2AB (2-aminobenzamide), a widely used fluorescent labels for glycosylation analysis. I think you will be impressed.
Check out the data sheet if you’re interested in glycan analysis by LC-MS too. It has examples of direct analysis of native, unlabeled glycans and charge-based quantitative glycan determination. Another example is the determination of glycosylation in a monoclonal antibody therapeutic by LC-MS. Variations in glycosylation can affect the drug’s safety and efficacy.
Stay tuned for Part II, for an overview of the webinar and techniques for glycan analysis. Don’t forget to add your questions or comments in the box below; we look forward to hearing from you.