Characterization of Site-Specific N-Glycopeptide Isoforms of α-1-Acid Glycoprotein from an Interlaboratory Study Using LC-MS/MS
| Autor: | Soo-Youn Lee, Junseok Kim, Seul Ki Jeong, Nari Seo, Seung Il Kim, Je-Yoel Cho, Gun Wook Park, Hyun Joo An, Myoung Jin Oh, Yong In Kim, Heeyoun Hwang, Sung Ho Yun, Young Ki Paik, Ki Na Yun, Su-yeon Kim, Jisook Park, Ju Yeon Lee, Hoi Keun Jeong, Kwang Hoe Kim, Jawon Seo, Chi Won Choi, Eun Sun Ji, Hyun Kyoung Lee, Jin Young Kim, Jong Sun Lim, Young Jin Choi, JongWon Kim, Jong Shin Yoo |
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| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Gene isoform Glycosylation α 1 acid glycoprotein Orbitrap Mass spectrometry Biochemistry law.invention 03 medical and health sciences law Tandem Mass Spectrometry Humans Protein Isoforms Sample preparation chemistry.chemical_classification Blood Specimen Collection Chromatography Binding Sites Chemistry Glycopeptides Reproducibility of Results General Chemistry Orosomucoid Glycopeptide 030104 developmental biology Glycoprotein Oxonium ion Chromatography Liquid |
| Zdroj: | Journal of proteome research. 15(12) |
| ISSN: | 1535-3907 |
| Popis: | 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 was25% 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. |
| Databáze: | OpenAIRE |
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