Site- and Structure-Specific Quantitative N-Glycoproteomics Using RPLC-pentaHILIC Separation and the Intact N-Glycopeptide Search Engine GPSeeker

Site- and structure-specific quantitative N-glycoproteomics characterization of differentially expressed N-glycosylation at the intact N-glycopeptide level with distinct chromatographic separation and structure-specific fragment ions has become possible with the recent development of RPLC-pentaHILIC 2DLC separation and use of the intact N-glycopeptide search engine GPSeeker. Here we provide a detailed protocol for this GPSeeker-centered structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. The protocols include sample preparation of a 1:1 mixture of light (-CH₃)₂ and heavy (-¹³CD₂H)₂ dimethylated intact N-glycopeptides from LO2 and HepG2 cells, RPLC-pentaHILIC 2DLC separation of the mixture, intact N-glycopeptide database search and identification using GPSeeker, and quantitation of differentially expressed intact N-glycopeptides using the quantitation module GPSeekerQuan. © 2019 by John Wiley & Sons, Inc.