| Budget Amount *help |
¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2005: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
This research was aimed at the development of a method for high-throughput and large-scale analysis of glycosylated proteins using the worm C.elegans as a source of complex protein mixture.
1.Large-scale indentification of the worm glycoproteins.
The worms, which were cultured in liquid medium, were disrupted by sonication and their proteins were separated into the soluble and insoluble fractions. Each protein fractions were dissolved with 7M guanidine-HCl solution buffered with 0.5 M Tris-HCl, reduced with dithiothreitol, and alkylated with iodo acetoamide. The proteins were digested with trypsin and the aliquots were separately applied to three types of lectin affinity chromatography columns, conA, wheat germ agglutinin (WGA), and the worm glectin-6 (GaL6). Obtained glycopeptides were further purified by hydrophilic interaction chromatography on Sepharose column. The glycopeptides were treated with N-glycopeptidase A in o-18 stable-isotope lebeled water (H_2^<18>O). The labeled peptide
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s were analyzed by 2D-LC-MS/MS shotgun proteome analysis system. Based on the MS/MS data, the peptides were identified by database searching using MASCOT as search engine and wormpep sequence dataset. Finally, total 830 proteins were identified as N-glycoproteins and their glycosylated sites were determined.
2. Large-scale quantification of the worm glycoproteins.
In order to develop a method for quantitative analysis of the glycoproteins, a reagent to introduce a mass-tag, which is differentially labeled with C-12/C-13 and N-14/N-15, was sellected and several reaction conditions were tested using chicken ovomucoid as a model glycoprotein. Ovomucoid was digested with trypsin and the resultant peptide mixture was separated into two fractions. One was lebeled with the reagent with light isotopes, and the other was with the reagent with heavy isotopes. The two fractions were mixed at some given ratio, and treated with glycopeptidase in o-18 lebeled water. The peptides were analyzed by nanoLC-MS/MS method and its glycopeptides were identified and relatively quantified. From the MS signal intensity, the glycopeptides could be relatively quantified at given mixd ratio. Now, using a complex protein mixture of the worm conA-bound peptides, conditions for overall procedures are testing. Less
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