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.