ポスター発表
- 第4日 5月18日(金) ポスター会場
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4P-01 PDF
付加位置特異的糖鎖不均一性解析法Glyco-RIDGE法の開発とpLNキャリア同定への応用
To analyze both peptide and glycan of glycopeptide, multiple fragmentation (MSn) analysis and interpretation of the resultant complex spectra are required. Ionization efficiency of glycopeptide is considerably lower compared with that of non-glycopeptide. MSn analysis further lowers its sensitivity. Therefore, we developed a method to assign both peptide sequence and glycan composition of glycopeptide without MS2 fragment information to improve the low assignment of site-specific glycan heterogeneity analysis. The method was named “Glyco-RIDGE”. In addition, to decrease sample complexity of glycopeptides, they were separated by hydrophilic interaction chromatography (HILIC) on an Amide-80 column. An aliquot of each glycopeptide fraction was treated with PNGase F in H218O and analyzed by LC/MS to identify the core peptides. Then, the released glycans were recovered and analyzed by MALDI MS after permethylation. The other aliquot was analyzed by LC/MS, where MS1 were acquired at high resolution (100,000 at m/z400) and HCD MS2 were measured in a data-dependent mode (2 intense ions / scan). From MS1 data, glycopeptide signals were selected by Glyco-RIDGE software as clusters. Glycopeptides were assigned by matching the observed accurate mass of glycopeptide and the calculated masses of core peptide + glycan in the list. Glycopeptides containing a long glycan (#Hex+HexNAc on a trimannosyl core ≥9) were presumed as polylactosamine (pLN)-carrying peptides.