| Budget Amount *help |
¥3,990,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥390,000)
Fiscal Year 2007: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2006: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
This research was aimed at the development of a method for high-throughput and large-scale quantitative analysis of glycosylated proteins which are contained in complex protein mixture such as tissue extracts and body fluids. The method for protein identification and quatitation is based on liquid chromatography / mass spectrometry (LC/MS) shotgun proteomic analysis technology. We developed mainly three parts of procedure in an overall approach.
1. Peptide preparation procedure. Protease digestion of a complex protein mixture results in a further complex peptide mixture Here, partial digestion significantly increases the complexity of the peptide fraction and thereby particular peptide speacies are decreased, which causes a lowering of the sensitivity of peptide identification. Thus, the conditions of peptide preparation were improved, i. e., (1) proteins were dissoled with 6M guanidine-HC1, reduced and alkylated, and dialyzed against a buffer containing 4M urea, and (2) the protein sol
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ution was treated with a protease, trypsin, after 2-fold dilution.
2. Large-scale identification and quantitation of N-linked glycoproteins by differential stable-isotope labeling. Two methods for the differential stable isotope labeling of peptide portion were tested, i. e., an enzymatic procedure using Peptide : N-glycanase (PNGase) and ^<18>O-labeled water (H_2^<18>O), and a chemical procedure using ^<13>C, ^<15>N-double labeled modifier (O-methylisourea) which is react with epsilon-amino group of Lys. Glycopeptides were captured by lectin (conA) affinity chromatography and purified by hydrophilic interaction chromatography (HIC) on Sepharose column. An aliquot of the glycopeptides fraction was treated with PNGase and H_2^<18>O, and another aliquot was treated by the same way using ordinary water (H_2^<18>O). Equimolar mixture of the two reaction products was analyzed by LC/MS/MS method. About 500 glycopeptides were identified and quantified at an average molar ratio of 0.80 +/- 0.14. By the same way, 150 glycopeptides were quantified at 0.98+/- 0.2 by the chemical modification approach.
3. An application of the accurate-mass approach for high sensitivity identification of glycopeptides. Based on the results of large-scale identification of glycoproteins of mouse liver, a list of accurate masses of core-peptide of glycopeptides was prepared. By comparing their accurate masses, about 70% of peptides (ca1, 100 peptides) were found to be distinguishable each other. This indicates a possibility of highly sensitive assignment of glycopeptides by accurate mass approach. Less
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