Protein glycosylation
is a family of posttranslational modifications
that play a crucial role in many biological pathways and diseases.
The enrichment and analysis of such a diverse family of modifications
are very challenging because of the number of possible glycan–peptide
combinations. Among the methods used for the enrichment of glycopeptides,
boronic acid never lived up to its promise. While most studies focused
on improving the affinity of the boronic acids to the sugars, we discovered
that the buffer choice is just as important for successful enrichment
if not more so. We show that an amine-less buffer allows for the best
glycoproteomic coverage, in human plasma and brain specimens, improving
total quantified glycopeptides by over 10-fold, and reaching 1598
N-linked glycopeptides in the brain and 737 in nondepleted plasma.
We speculate that amines compete with the glycans for boronic acid
binding, and therefore the elimination of them improved the method
significantly.