Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation

• Published in: Scientific reports 2016-02, Vol.6 (1), p.21175-21175, Article 21175
• Main Authors: Park, Gun Wook, Kim, Jin Young, Hwang, Heeyoun, Lee, Ju Yeon, Ahn, Young Hee, Lee, Hyun Kyoung, Ji, Eun Sun, Kim, Kwang Hoe, Jeong, Hoi Keun, Yun, Ki Na, Kim, Yong-Sam, Ko, Jeong-Heon, An, Hyun Joo, Kim, Jae Han, Paik, Young-Ki, Yoo, Jong Shin
• Format: Article
• Language: English
• Subjects: 631/1647/296; 631/1647/48; Algorithms; Automation; Automation, Laboratory; Blood Proteins - metabolism; Carcinoma, Hepatocellular - blood; Carcinoma, Hepatocellular - metabolism; Glycopeptides; Glycopeptides - chemistry; Glycopeptides - metabolism; Glycoproteins; Glycoproteins - chemistry; Glycoproteins - metabolism; Glycosylation; Heterogeneity; Humanities and Social Sciences; Humans; Liver Neoplasms - blood; Liver Neoplasms - metabolism; Mapping; Mass spectrometry; Mass spectroscopy; multidisciplinary; Proteome; Proteomes; Proteomics - instrumentation; Proteomics - methods; Quantitation; Reproducibility of Results; Science
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep21175

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.