title: Online coupling of hydrophilic interaction/strong cation exchange/reversed-phase liquid chromatography with porous graphitic carbon liquid chromatography for simultaneous proteomics and N-glycomics analysis.
abstract: In this study we developed a fully automated three-dimensional (3D) liquid chromatography methodology-comprising hydrophilic interaction separation as the first dimension, strong cation exchange fractionation as the second dimension, and low-pH reversed-phase (RP) separation as the third dimension-in conjunction downstream with additional complementary porous graphitic carbon separation, to capture non-retained hydrophilic analytes, for both shotgun proteomics and N-glycomics analyses. The performance of the 3D system alone was benchmarked through the analysis of the total lysate of Saccharomyces cerevisiae, leading to improved hydrophilic peptide coverage, from which we identified 19% and 24% more proteins and peptides, respectively, relative to those identified from a two-dimensional hydrophilic interaction liquid chromatography and low-pH RP chromatography (HILIC-RP) system over the same mass spectrometric acquisition time; consequently, the 3D platform also provided enhanced proteome and protein coverage. When we applied the integrated technology to analyses of the total lysate of primary cerebellar granule neurons, we characterized a total of 2201 proteins and 16,937 unique peptides for this primary cell line, providing one of its most comprehensive datasets. Our new integrated technology also exhibited excellent performance in the first N-glycomics analysis of cynomolgus monkey plasma; we successfully identified 122 proposed N-glycans and 135 N-glycosylation sites from 122 N-glycoproteins, and confirmed the presence of 38 N-glycolylneuraminic acid-containing N-glycans, a rare occurrence in human plasma, through tandem mass spectrometry for the first time.
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Henry C H
authors.initials: HC
authors.lastname: Law
authors.affiliation: Institute of New Drug Research and Guangdong Province Key Laboratory of Pharmacodynamic Constituents of Traditional Chinese Medicine, Jinan University College of Pharmacy, Guangzhou 510632, China. Electronic address: zaijunzhang@163.com.
authors.forename: Zaijun
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Herman C
authors.initials: HC
authors.lastname: Lam
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Quan
authors.initials: Q
authors.lastname: Quan
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China.
authors.forename: Guohui
authors.initials: G
authors.lastname: Li
authors.affiliation: Department of Chemistry, The University of Hong Kong, Hong Kong, China. Electronic address: ivankchu@hku.hk.
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Chu
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002460:Cell Line; D002531:Cerebellum / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D054794:Glycomics / Q000379:methods; D006023:Glycoproteins / Q000097:blood; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008252:Macaca fascicularis; D008297:Male; D009474:Neurons / Q000737:chemistry; D010455:Peptides / Q000032:analysis*; D016062:Porosity; D011506:Proteins / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D006023:Glycoproteins; D010455:Peptides; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins
keywords: HILIC–SCX–RP; N-Glycans; N-Glycoproteins; Proteomics; Three-dimensional liquid chromatography