title: Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS.
abstract: 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.
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.
authors.forename: Ju Yeon
authors.initials: JY
authors.lastname: Lee
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.|Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Hyun Kyoung
authors.initials: HK
authors.lastname: Lee
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.|Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Gun Wook
authors.initials: GW
authors.lastname: Park
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.
authors.forename: Heeyoun
authors.initials: H
authors.lastname: Hwang
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.|Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Hoi Keun
authors.initials: HK
authors.lastname: Jeong
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.|Department of Chemistry, Sogang University , Seoul 04107, Republic of Korea.
authors.forename: Ki Na
authors.initials: KN
authors.lastname: Yun
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.|Department of Chemistry, Hannam University , Daejeon 34430, Republic of Korea.
authors.forename: Eun Sun
authors.initials: ES
authors.lastname: Ji
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.|Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Kwang Hoe
authors.initials: KH
authors.lastname: Kim
authors.affiliation: Department of Biomedical Systems Engineering, Korea Polytechnics , Gyeonggi 13590, Republic of Korea.
authors.forename: Jun Seok
authors.initials: JS
authors.lastname: Kim
authors.affiliation: New Drug Development Center, Osong Medical Innovation Foundation , Cheongju 28160, Republic of Korea.
authors.forename: Jong Won
authors.initials: JW
authors.lastname: Kim
authors.affiliation: Drug & Disease Target Group, Korea Basic Science Institute , Daejeon 34133, Republic of Korea.
authors.forename: Sung Ho
authors.initials: SH
authors.lastname: Yun
authors.affiliation: Drug & Disease Target Group, Korea Basic Science Institute , Daejeon 34133, Republic of Korea.
authors.forename: Chi-Won
authors.initials: CW
authors.lastname: Choi
authors.affiliation: Drug & Disease Target Group, Korea Basic Science Institute , Daejeon 34133, Republic of Korea.
authors.forename: Seung Il
authors.initials: SI
authors.lastname: Kim
authors.affiliation: Yonsei Proteome Research Center, Yonsei University , Seoul 03722, Republic of Korea.
authors.forename: Jong-Sun
authors.initials: JS
authors.lastname: Lim
authors.affiliation: Yonsei Proteome Research Center, Yonsei University , Seoul 03722, Republic of Korea.
authors.forename: Seul-Ki
authors.initials: SK
authors.lastname: Jeong
authors.affiliation: Yonsei Proteome Research Center, Yonsei University , Seoul 03722, Republic of Korea.
authors.forename: Young-Ki
authors.initials: YK
authors.lastname: Paik
authors.affiliation: Department of Laboratory & Genetics, Samsung Medical Center, Sungkyunkwan University of Medicine , Seoul 06351, Republic of Korea.|Department of Clinical Pharmacology and Therapeutics, Samsung Medical Center , Seoul 06351, Republic of Korea.
authors.forename: Soo-Youn
authors.initials: SY
authors.lastname: Lee
authors.affiliation: Samsung Biomedical Research Institute, Samsung Medical Center, Sungkyunkwan University School of Medicine , Seoul 06351, Republic of Korea.
authors.forename: Jisook
authors.initials: J
authors.lastname: Park
authors.affiliation: Department of Clinical Research Supporting Team, Clinical Research Institute, Samsung Medical Center , Seoul 06351, Republic of Korea.
authors.forename: Su Yeon
authors.initials: SY
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, Republic of Korea.
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Choi
authors.affiliation: Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, Republic of Korea.
authors.forename: Yong-In
authors.initials: YI
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, Republic of Korea.
authors.forename: Jawon
authors.initials: J
authors.lastname: Seo
authors.affiliation: Department of Biochemistry, BK21 PLUS Program for Creative Veterinary Science Research and Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University , Seoul 08826, Republic of Korea.
authors.forename: Je-Yoel
authors.initials: JY
authors.lastname: Cho
authors.affiliation: Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Myoung Jin
authors.initials: MJ
authors.lastname: Oh
authors.affiliation: Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Nari
authors.initials: N
authors.lastname: Seo
authors.affiliation: Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Hyun Joo
authors.initials: HJ
authors.lastname: An
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.
authors.forename: Jin Young
authors.initials: JY
authors.lastname: Kim
authors.affiliation: Biomedical Omics Group, Korea Basic Science Institute , Ochang 28119, Republic of Korea.|Graduate School of Analytical Science and Technology, Chungnam National University , Daejeon 34134, Republic of Korea.
authors.forename: Jong Shin
authors.initials: JS
authors.lastname: Yoo
mesh_terms: D001665:Binding Sites; D001800:Blood Specimen Collection; D002853:Chromatography, Liquid; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D009961:Orosomucoid / Q000737:chemistry*; D020033:Protein Isoforms / Q000032:analysis*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D006020:Glycopeptides; D009961:Orosomucoid; D020033:Protein Isoforms
keywords: PTM (post translational modification); interlaboratory study; isoforms; site-specific N-glycopeptide