发明申请
- 专利标题: In-Gel Tagging And In-Gel Digestion For Phosphoproteins Analysis And Phosphorylation Site Identification
- 专利标题(中): 凝胶标记和凝胶消化磷蛋白分析和磷酸化位点鉴定
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申请号: US11574660申请日: 2004-10-08
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公开(公告)号: US20070287169A1公开(公告)日: 2007-12-13
- 发明人: Yeong Ahn , Jong Yoo , Jae Lee , Jin Kim , Kun Cho
- 申请人: Yeong Ahn , Jong Yoo , Jae Lee , Jin Kim , Kun Cho
- 申请人地址: KR Daejeon-shi 305-333
- 专利权人: KOREA BASIC SCIENCE INSTITUTE
- 当前专利权人: KOREA BASIC SCIENCE INSTITUTE
- 当前专利权人地址: KR Daejeon-shi 305-333
- 优先权: KR10-2004-0070651 20040904
- 国际申请: PCT/KR04/02577 WO 20041008
- 主分类号: C12P21/06
- IPC分类号: C12P21/06 ; C07K14/00
摘要:
The present invention relates to a method for phosphorylation site-specific labeling of phosphoproteome with a site-specific tagging reagent and analyzing of the resulting labeled one, more especially, a method for in-situ tagging of phosphorylation sites of phosphoproteins retained in polymeric gel with a nucleophilic tagging reagent. It also relates a method for generating new proteolytic cleavable sites at formerly phosphorylation sites by a proper choice of a nucleophilic tagging reagent. It also relates to a method for phosphopeptides analysis and phosphorylation site identification by in-gel digestion of the previously in-gel tagged proteins and subsequent mass analysis of the resulting peptides. The invention provides in-gel chemical tagging method for phosphoaminoacid residue of phosphoproteins retained in polymeric gel matrix. Phosphoprotein can be immobilized into gel matrix by a variety of methods such as gel electrophoresis. The immobilized phosphoproteins are retained in gel matrix during tagging reaction to phosphorylated aminoacid residue of phosphoproteins, and the resulting tagged proteins are also retained in gel matrix till following purification steps like washing of the tagging reagents are accomplished. The tagged proteins is digested by protease, and the resulting digested peptides is released from gel into solution and applied for peptide mass analysis.