10.1021/ac400315n.s003
Jing Liu
Jing
Liu
Fangjun Wang
Fangjun
Wang
Hui Lin
Hui
Lin
Jun Zhu
Jun
Zhu
Yangyang Bian
Yangyang
Bian
Kai Cheng
Kai
Cheng
Hanfa Zou
Hanfa
Zou
Monolithic Capillary Column Based Glycoproteomic Reactor for High-Sensitive Analysis of N‑Glycoproteome
American Chemical Society
2013
Monolithic Capillary Column
capillary column
detection
glycopeptide
cm
novel glycoproteomic reactor
deglycosylation
site
sample
protein
mapping
enrichment
2013-03-05 00:00:00
Journal contribution
https://acs.figshare.com/articles/journal_contribution/Monolithic_Capillary_Column_Based_Glycoproteomic_Reactor_for_High_Sensitive_Analysis_of_N_Glycoproteome/2437534
Despite 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 ∼10<sup>4</sup> HeLa cells.