Journal
ANALYTICAL CHEMISTRY
Volume 85, Issue 5, Pages 2847-2852Publisher
AMER CHEMICAL SOC
DOI: 10.1021/ac400315n
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Funding
- Creative Research Group Project by NSFC [21021004]
- Scientific Research Equipment Development Projects of CAS [P020090520]
- China State Key Basic Research Program Grant [2013CB911202, 2012CB910601, 2012CB910101]
- Analytical Method Innovation Program of MOST [2012IM030900]
- Hundred Talent Young Scientist Program by DICP
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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 similar to 10(4) HeLa cells.
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