Fast solid-phase extraction of N-linked glycopeptides by amine-functionalized mesoporous silica nanoparticles

Selective enrichment is a crucial step before the mass spectrometric analysis of glycoproteins. A new approach using 3-aminopropyltriethoxysilane (APTES)-functionalized mesoporous silica materials (SBA-15) was reported to enrich the glycoproteins. Selective extraction of glycopeptides was achieved through coupling the oxidized glycan chains on the glycopeptides with the amine groups on SBA-15 through a reductive amination reaction, then the captured glycopeptides were detached from the SBA-15 for the following MS analysis using the enzyme PNGase F. Because the mesoporous material has a confinement effect, the efficiency of enrichment and enzymatic deglycosylation was improved dramatically. The coupling time was shortened from 4 hours to 1 hour, and the deglycosylation time was greatly shortened from 6 hours to 3 hours. This approach was successfully applied to profile the N-glyco-proteome of human colorectal cancer serum. 84 N-linked glycosylation sites from 56 N-linked glyco-proteins were identified from as little as 5 mu L serum.