Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS

Ju Yeon Lee, Hyun Kyoung Lee, Gun Wook Park, Heeyoun Hwang, Hoi Keun Jeong, Ki Na Yun, Eun Sun Ji, Kwang Hoe Kim, Jun Seok Kim, Jong Won Kim, Sung Ho Yun, Chi Won Choi, Seung Il Kim, Jong Sun Lim, Seul Ki Jeong, Young Ki Paik, Soo Youn Lee, Jisook Park, Su Yeon Kim, Young Jin ChoiYong In Kim, Jawon Seo, Je Yoel Cho, Myoung Jin Oh, Nari Seo, Hyun Joo An, Jin Young Kim, Jong Shin Yoo

Research output: Contribution to journalArticle

17 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)4146-4164
Number of pages19
JournalJournal of Proteome Research
Issue number12
Publication statusPublished - 2016 Dec 2


All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Chemistry(all)

Cite this

Lee, J. Y., Lee, H. K., Park, G. W., Hwang, H., Jeong, H. K., Yun, K. N., Ji, E. S., Kim, K. H., Kim, J. S., Kim, J. W., Yun, S. H., Choi, C. W., Kim, S. I., Lim, J. S., Jeong, S. K., Paik, Y. K., Lee, S. Y., Park, J., Kim, S. Y., ... Yoo, J. S. (2016). Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS. Journal of Proteome Research, 15(12), 4146-4164.