Amine Chemistry Method for Selective Enrichment of N‑Linked Glycopeptides for Glycoproteomics Analysis

An amine chemistry method was developed
for the extraction of N-glycopeptides
using amine-functionalized beads for glycoproteomics analysis. Two
reductive amination reactions between primary amine and aldehyde were
employed in this approach. The first one was to block the primary
amines in the peptides by addition of formaldehyde and sodium cyanoborohydride
into the peptide sample, and the second one was to couple the glycopeptides
onto solid phase beads by incubating the glycopeptides containing
aldehyde groups (oxidized by periodate) with the amine-functionalized
beads in the presence of sodium cyanoborohydride. It was demonstrated
that the blocking of primary amines in the peptides by the first reductive
amination reaction prior to the periodate oxidation made the amine
chemistry method very efficient and sensitive. This new method was
validated by analysis of glycoprotein standards as well as proteome
samples. It was found that this new method led to significant increase
in the identification of N-glycosites compared with the conventional
hydrazide chemistry method.