An optimized glycopeptide enrichment method – it’s all about the sauce

Protein glycosylation is a family of post translational modifications (PTMs) that play a crucial role in cellular and tissue biology, and whose role in disease is unraveled with every new finding. Enrichment and analysis of such a diverse family of modification is very challenging, due to the number of possible glycan-peptide combinations. Among the methods used for enrichment of glycopeptides, boronic acid never lived up to its promise. While most studies focused on improving the affinity of the sugars to boronic acid, we discovered that buffer choice is as important for successful enrichment if not more. We show that amine-less buffer allows for the best enrichment results in human plasma and brain specimens. We speculate that amines compete with the glycans for boronic acid binding, therefore eliminating them improved the method significantly.