Gun Wook Park 1,2, Jin Young Kim 1, Ju Yeon Lee 1, Young-Mook Kang 1, Heeyoun Hwang 1, Hyun Kyoung Lee 1,2, Eun Sun Ji 1,3, Kwang Hoe Kim 1,2, Hoi Keun Jeong 1,2, Ki Na Yun 1,3, Yong-Sam Kim 4, Jeong-Heon Ko 4, and Jong Shin Yoo 1,2
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 Chemistry, Sogang University, Seoul, Republic of Korea
4 Cancer Biomarkers Development Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea
We have developed Integrated GlycoProteomeAnalyzer (GPA) for high throughput analysis of N-and O-glycoproteome, which combines methods for tandem mass spectrometry with a database search and algorithmic suite. We created novel scoring algorithms with calculation of false discovery rate (FDR) and label-free quantification method using the combined intensities of top three isotope peaks at three highest MS spectral points..
All glycoprotein samples were digested with trypsin. Then, those site-specific N-glycopeptides were enriched by hydrophobic interaction liquid chromatography and analyzed by nano-reversed-phase liquid chromatography coupled to tandem mass spectrometry with both HCD and CID-MS/MS fragmentation. The resultant LC/MS/MS data were then automatically analyzed using GPA: tryptic N-glycopeptides were identified against the database from Uniprot, quantified using the 3TIQ, and finally compared between multiple samples. In GPA, all amino acid sequence and N-and O-glycosylation site information were obtained from the Uniprot database. From the Swiss-Prot accession number of human protein, GPA program automatically construct N-glycopeptide database for human plasma sample.
GPA has been designed to easily handle high-throughput glycoproteomic data with a graphical user interface. GPA software is demonstrated on website
(https://www.igpa.kr/). Additionally, O-GPA, newly developed search engine, allows direct analysis of site-specific O-glycopeptides from glycoprotein mixtures using the O-GPA-DB from Uniprot with estimated FDR ≤ 1%. O-GPA is capable of automatic identification and quantification of O-glycopeptides using HCD and ETD-MS/MS spectra.
Park. et al, Scientific Reports. (2016) 6:21175, DOI: 10.1038/srep21175.