Integrated GlycoProteome Analyzer (I-GPA) for Automated Identification and Quantitation of Site-Specific N-Glycosylation.
1 Department of Mass Spectrometry, Korea Basic Science Institute, Ochang, Republic of Korea.
2 Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon, Republic of Korea.
3 Department of Biomedical Science, Cheongju University, Cheongju, Republic of Korea.
4 Department of Chemistry, Hannam University, Daejeon, Republic of Korea.
5 Department of Chemistry, Sogang University, Seoul, Republic of Korea.
6 Cancer Biomarkers Development Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea.
7 Department of Food Nutrition, Chungnam National University, Daejeon, Republic of Korea.
8 Yonsei Proteome Research Center and Department of Integrated OMICS for Biomedical Science, and Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea.
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.