Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.
Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.
复制标题
DOI：
10.1038/srep21175
复制
发表时间：
2016-02-17
影响因子：
4.6
通讯作者：
Yoo JS
中科院分区：
文献类型：
--
作者：
Park GW;Kim JY;Hwang H;Lee JY;Ahn YH;Lee HK;Ji ES;Kim KH;Jeong HK;Yun KN;Kim YS;Ko JH;An HJ;Kim JH;Paik YK;Yoo JS
人类糖蛋白在每个N-糖位点上表现出巨大的异质性，但很少有研究试图全面表征位点特异性结构特征。我们已经开发了集成糖蛋白质组分析仪（I-GPA），包括复杂的N-糖蛋白质组的映射系统，它结合了串联质谱与数据库搜索和算法套件的方法。使用我们构建的N-糖肽数据库，我们使用诱饵糖肽创建了新的评分算法，其中标准α1-酸性糖蛋白中的95个N-糖肽被鉴定为0%假阳性，得到与手动验证相同的结果。此外，首次开发了自动化无标记定量方法，该方法利用三个最高MS光谱点处前三个同位素峰的组合强度。通过从已知含有高度糖基化蛋白的人血浆样本中自动识别FDR ≤ 1%的619个位点特异性N-糖肽，并同时定量598个N-糖肽，证明了I-GPA的有效性。因此，I-GPA平台可以在复杂N-糖蛋白质组的高通量作图方面取得重大突破，这可以应用于生物标志物的发现和正在进行的全球人类蛋白质组计划。
Human glycoproteins exhibit enormous heterogeneity at each N-glycosite, but few studies have attempted to globally characterize the site-specific structural features. We have developed Integrated GlycoProteome Analyzer (I-GPA) including mapping system for complex N-glycoproteomes, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. Using an N-glycopeptide database that we constructed, we created novel scoring algorithms with decoy glycopeptides, where 95 N-glycopeptides from standard α1-acid glycoprotein were identified with 0% false positives, giving the same results as manual validation. Additionally automated label-free quantitation method was first developed that utilizes the combined intensity of top three isotope peaks at three highest MS spectral points. The efficiency of I-GPA was demonstrated by automatically identifying 619 site-specific N-glycopeptides with FDR ≤ 1%, and simultaneously quantifying 598 N-glycopeptides, from human plasma samples that are known to contain highly glycosylated proteins. Thus, I-GPA platform could make a major breakthrough in high-throughput mapping of complex N-glycoproteomes, which can be applied to biomarker discovery and ongoing global human proteome project.