Glycoprotein conformations are complex and heterogeneous. Currently, site-specific characterization of glycopeptides is a challenge. We sought to establish an efficient method of N-glycoprotein characterization using mass spectrometry (MS). Using alpha-1-acid glycoprotein (AGP) as a model N-glycoprotein, we identified its tryptic N-glycopeptides and examined the data reproducibility in seven laboratories running different LC-MS/MS platforms. We used three test samples and one blind sample to evaluate instrument performance with entire sample preparation workflow. 165 site-specific N-glycopeptides representative of all N-glycosylation sites were identified from AGP 1 and AGP 2 isoforms. The glycopeptide fragmentations by collision-induced dissociation or higher-energy collisional dissociation (HCD) varied based on the MS analyzer. Orbitrap Elite identified the greatest number of AGP N-glycopeptides, followed by Triple TOF and Q-Exactive Plus. Reproducible generation of oxonium ions, glycan-cleaved glycopeptide fragment ions, and peptide backbone fragment ions was essential for successful identification. Laboratory proficiency affected the number of identified N-glycopeptides. The relative quantities of the 10 major N-glycopeptide isoforms of AGP detected in four laboratories were compared to assess reproducibility. Quantitative analysis showed that the coefficient of variation was <25% for all test samples. Our analytical protocol yielded identification and quantification of site-specific N-glycopeptide isoforms of AGP from control and disease plasma sample.
Bibliographical noteFunding Information:
This work was supported by the Korea Health Technology RandD Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health and Welfare, Republic of Korea (grant number: HI13C2098), by the Creative Allied Project (CAP) through the National Research Council of Science and Technology (grant number: NTM2371511), by the research program through the Korea Basic Science Institute (grant numbers: K36110), by the Bio-Synergy Research Project (grant number: NRF-2014M3A9C4066461) of the Ministry of Science, ICT and Future Planning through the National Research Foundation, and by the National Research Foundation of Korea Grant funded by the Korea Government (MSIP) (2016, RandD Equipment Engineer Education Program, 2014R1A6A9064166).
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