title: Highly efficient N-glycoproteomic sample preparation by combining C(18) and graphitized carbon adsorbents.
abstract: Conventional N-glycoproteome analysis usually applies C18 reversed-phase (RP) adsorbent for sample purification, which will lead to unavoidable sample loss due to the high hydrophilicity of N-glycopeptides. In this study, a porous graphitized carbon (PGC) absorbent was combined with a C18 adsorbent for N-glycopeptide purification in comprehensive N-glycoproteome analysis based on the hydrophobic and polar interactions between carbon and N-glycans. It was observed that the small hydrophilic N-glycopeptides that cannot retain onto C18 adsorbent can be captured by the graphitized carbon, while the large hydrophobic N-glycopeptides that cannot retain onto the graphitized carbon can be feasibly captured by the C18 adsorbent. Comparing with sample purification by using C18 adsorbent only, 28.5 % more N-glycopeptides were identified by combining both C18 and PGC adsorbents. The C18-PGC strategy was further applied for both sample purification and pre-fractionation of a complex protein sample from HeLa cell. After hydrophilic interaction chromatography enrichment, 1,484 unique N-glycopeptides with 1,759 unique N-glycosylation sites were finally identified.
authors.affiliation: Key Laboratory of Separation Sciences for Analytical Chemistry, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences (CAS), 116023, Dalian, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Fangjun
authors.initials: F
authors.lastname: Wang
authors.forename: Jun
authors.initials: J
authors.lastname: Zhu
authors.forename: Jiawei
authors.initials: J
authors.lastname: Mao
authors.forename: Zheyi
authors.initials: Z
authors.lastname: Liu
authors.forename: Kai
authors.initials: K
authors.lastname: Cheng
authors.forename: Hongqiang
authors.initials: H
authors.lastname: Qin
authors.forename: Hanfa
authors.initials: H
authors.lastname: Zou
mesh_terms: D002244:Carbon / Q000737:chemistry* / Q000378:metabolism; D002853:Chromatography, Liquid; D056148:Chromatography, Reverse-Phase; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D006031:Glycosylation; D006108:Graphite / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D013048:Specimen Handling; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006020:Glycopeptides; D020543:Proteome; D002244:Carbon; D006108:Graphite