iTRAQ plus ¹⁸O: A new technique for target glycoprotein analysis

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• 作者：Shu Zhang^a ; ^c ; Xiaohui Liu^b ; ^c ; Xiaonan Kang^c ; Chun Sun^c ; Haojie Lu^b ; ^c ; ^{luhaojie@fudan.edu.cn} ; Pengyuan Yang^b ; ^c ; Yinkun Liu^a ; ^c ; ^{liu.yinkun@zs-hospital.sh.cn}
• 关键词：N-glycosylation ; Quantification ; iTRAQ plus 18O ; Haptoglobin ; Liver diseases
• 刊名：Talanta
• 出版年：2012
• 期刊代码：106_00399140
• 类别：ch
• 出版时间：15 March, 2012
• 卷：91
• 期：Complete
• 页码：122-127
• 文件大小：843 K

摘要

A novel strategy combining iTRAQ with ¹⁸O stable isotope labeling (iTRAQ plus ¹⁸O) was established to identify N-glycosylation site, quantify the glycopeptides and non-glycosylated peptides, and obtain N-glycosylation site ratio on the target glycoprotein. In this approach, all peptides of four biological samples are labeled with four iTRAQ reagents in parallel, followed by PNGase F catalyzed labeling of N-glycosylation sites with H₂¹⁶O and H₂¹⁸O. Two sample groups are labeled with H₂¹⁶O and the other two are labeled with H₂¹⁸O. After the modification of MS precursor ion isolation window, tagged peptides are identified by LC-MS/MS, both glycopeptides and non-glycopeptides are quantified simultaneously using ProteinPilot鈩?Software. With four samples to be maximally analyzed in parallel, this workflow supports accurate identification and quantification of glycopeptides in a site-specific fashion. Furthermore, N-glycosylation site ratios on serum haptoglobin (Hp) 尾 chain in healthy individuals as well as patients with hepatitis B virus (HBV), liver cirrhosis (LC) and hepatocellular carcinoma (HCC) were quantified to validate the novel 鈥榠TRAQ plus ¹⁸O鈥?method. Glycosite ratios of VVLHPN#YSQVDIGLIK were observed to change significantly in HCC patients compared with LC and HBV patients. This novel approach supports the screening of the target glycoproteins as biomarkers in clinical application.