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

Abstract 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 (‐CH 3 ) 2 and heavy (‐ 13 CD 2 H) 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.