成果報告書詳細
管理番号20130000000346
タイトル*平成24年度中間年報 「環境・医療分野の国際研究開発・実証プロジェクト フランスにおける国際共同研究開発・実証事業/OnーDemand Multiーmixed Protein Quantification Kitの技術開発」
公開日2014/8/20
報告書年度2012 - 2012
委託先名国立大学法人東北大学 株式会社Proteomedix Frontiers
プロジェクト番号P11017
部署名バイオテクノロジー・医療技術部
和文要約
英文要約Title: Japan-France collaborative industrial R&D demonstration programme. Technology development of an On-demand Multi-mixed Protein Quantification Kit (FY2012-FY2013) FY2012 Annual Report

The purpose of this project is to develop a LC-MS/MS-based technology for a next-generation kit allowing mass-scale simultaneous and absolute quantification of many proteins by a combination of molecular biology and quantitative targeted absolute proteomics (QTAP). A key of the quantification technology is to use stable isotope-labeled synthetic peptides as internal standards. However, the synthetic peptides limit the possible number of proteins to be quantified because of the high cost and the complicated simultaneous handling of many peptides. This project tries to overcome the limitation of synthetic peptides by producing stable isotope-labeled artificial protein containing internal standard peptides in tandem. In step 1, we selected the trypsin-digested sequences of target peptides to be quantified by using our patented in silico peptide selection criteria. In step2, we designed and synthesized cDNA construct of the series of internal standard peptides in tandem, in which two types of quantification tag peptides and purification tag peptides were added at amino-terminus and carboxyl-terminus. In step3, we produced a package of the stable-isotope labeled internal reference protein by using in vitro cell free system and E. coli protein synthesis system. In this protein synthesizing step, the yield, purity, stable-isotope labeling efficacy, and stability of the synthesized protein are critical problems for commercialization. We found that yield of protein synthesis by the cell free system depends on the physicochemical characteristics of the protein. In order to obtain a full-length protein with high purity, we have established the sequential two-step purification with both N- and C-terminus tags and two types of the corresponding affinity columns. The absolute amounts of trypsin-digested quantification tag peptides at N-terminus and C-terminus were equal, confirming that the full length form of synthesized protein can be highly purified. In terms of the efficiency of stable isotope labeling, the contamination of peptide consisting of native type amino acid in the stable isotope labeled peptide was less than 1 %. We examined several reagents for the protein stabilization after the synthesis. In step4, we determined the LC-MS/MS conditions for each peptide after trypsin digestion. The concentrated elution of many peptides at a similar retention and the too early and too late elution cause the lower sensitivity. This can be prevented by arranging the peptide elution based on the prediction of LC-elution time. The optimized protocol can proceed to validate the kit for several different materials including human tissues and cultured cells in a collaboration consortium of Bertin Pharma and University of Paris in French side. We also have investigated the demands of the quantification kits in the fields of drug development and determined the lineup of the kits which are targeted at the preclinical and clinical stages.
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