title: Monolithic capillary column based glycoproteomic reactor for high-sensitive analysis of N-glycoproteome.
abstract: 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(4) HeLa cells.
authors: CAS Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors: Jing
authors: J
authors: Liu
authors: Fangjun
authors: F
authors: Wang
authors: Hui
authors: H
authors: Lin
authors: Jun
authors: J
authors: Zhu
authors: Yangyang
authors: Y
authors: Bian
authors: Kai
authors: K
authors: Cheng
authors: Hanfa
authors: H
authors: Zou
mesh_terms: D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006367:HeLa Cells; D006735:Horseradish Peroxidase / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D011487:Protein Conformation; D059748:Proteolysis; D040901:Proteomics / Q000295:instrumentation*; D014357:Trypsin / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D006735:Horseradish Peroxidase; D014357:Trypsin