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

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• 作者：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 Choi ; Yong-In Kim ; Jawon Seo ; Je-Yoel Cho ; Myoung Jin Oh ; Nari Seo ; Hyun Joo An ; Jin Young Kim ; Jong Shin Yoo
• 刊名：Journal of Proteome Research
• 出版年：2016
• 出版时间：December 2, 2016
• 年：2016
• 卷：15
• 期：12
• 页码：4146-4164
• 全文大小：1519K
• ISSN：1535-3907

文摘

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.