title: Centrifugation assisted microreactor enables facile integration of trypsin digestion, hydrophilic interaction chromatography enrichment, and on-column deglycosylation for rapid and sensitive N-glycoproteome analysis.
abstract: Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
authors.affiliation: CAS Key Lab of Separation Sciences for Analytical Chemistry, National Chromatographic Research and Analysis Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.forename: Rui
authors.initials: R
authors.lastname: Chen
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Bo
authors.initials: B
authors.lastname: Xu
authors.forename: Zhimou
authors.initials: Z
authors.lastname: Guo
authors.forename: Xinmiao
authors.initials: X
authors.lastname: Liang
authors.forename: Mingliang
authors.initials: M
authors.lastname: Ye
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000595:Amino Acid Sequence; D002498:Centrifugation; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007074:Immunoglobulin G / Q000032:analysis*; D057230:Limit of Detection; D008969:Molecular Sequence Data; D059748:Proteolysis; D020543:Proteome / Q000032:analysis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
chemical_list: D000097:Acetonitriles; D006020:Glycopeptides; D007074:Immunoglobulin G; D020543:Proteome; D014269:Trifluoroacetic Acid; D014357:Trypsin; C032159:acetonitrile