ac400315n_si_003.pdf (40.6 kB)
Monolithic Capillary Column Based Glycoproteomic Reactor for High-Sensitive Analysis of N‑Glycoproteome
journal contribution
posted on 2013-03-05, 00:00 authored by Jing Liu, Fangjun Wang, Hui Lin, Jun Zhu, Yangyang Bian, Kai Cheng, Hanfa ZouDespite the importance of protein N-glycosylation in
a series of
biological processes, in-depth characterization of protein glycosylation
is still a challenge due to the high complexity of biological samples
and the lacking of highly sensitive detection technologies. We developed
a monolithic capillary column based glycoproteomic reactor enabling
high-sensitive mapping of N-glycosylation sites from minute amounts
of sample. Unlike the conventional proteomic reactors with only strong-cation
exchange or hydrophilic-interaction chromatography columns, this novel
glycoproteomic reactor was composed of an 8 cm long C12 hydrophobic
monolithic capillary column for protein digestion and a 6 cm long
organic–silica hybrid hydrophilic monolithic capillary column
for glycopeptides enrichment and deglycosylation, which could complete
whole-sample preparation including protein purification/desalting,
tryptic digestion, enrichment, and deglycosylation of glycopeptides
within about 3 h. The developed reactor exhibited high detection sensitivity
in mapping of N-glycosylation sites by detection limit of horseradish
peroxidase as low as 2.5 fmol. This reactor also demonstrated the
ability in complex sample analysis, and in total, 486 unique N-glycosylation
sites were reliably mapped in three replicate analyses of a protein
sample extracted from ∼104 HeLa cells.