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    In-gel tagging and in-gel digestion for phosphoproteins analysis and phosphorylation site identification
    1.
    发明授权
    In-gel tagging and in-gel digestion for phosphoproteins analysis and phosphorylation site identification 失效
    凝胶内标记和凝胶消化，用于磷酸化蛋白分析和磷酸化位点鉴定

    公开(公告)号：US07629744B2

    公开(公告)日：2009-12-08

    申请号：US11574660

    申请日：2004-10-08

    申请人： Yeong Hee Ahn ,  Jong Shin Yoo ,  Jae Yong Lee ,  Jin Young Kim ,  Kun Cho

    发明人： Yeong Hee Ahn ,  Jong Shin Yoo ,  Jae Yong Lee ,  Jin Young Kim ,  Kun Cho

    IPC分类号： H01J17/26

    CPC分类号： G01N33/6842 ,  G01N33/6848

    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.

    摘要翻译： 本发明涉及用位点特异性标记试剂对磷酸化蛋白质进行磷酸化位点特异性标记的方法，并分析得到的标记物，更具体地说，一种用于原位标记保留在聚合物凝胶中的磷酸化蛋白的磷酸化位点的方法， 亲核标记试剂。 它还涉及通过适当选择亲核标记试剂在以前的磷酸化位点产生新的蛋白水解可切割位点的方法。 它还涉及通过凝胶内消化先前凝胶内标记蛋白的磷酸肽分析和磷酸化位点鉴定的方法以及随后对所得肽的质量分析。 本发明提供了凝胶化学标记方法，用于保留在聚合物凝胶基质中的磷酸蛋白的磷酸氨基酸残基。 磷酸蛋白可以通过各种方法如凝胶电泳固定在凝胶基质中。 在磷酸化蛋白质的磷酸化氨基酸残基的标签反应期间，将固定的磷酸蛋白质保留在凝胶基质中，并且所得到的标记蛋白质也保留在凝胶基质中，直到完成标记试剂的洗涤之后的纯化步骤。 标记的蛋白质被蛋白酶消化，并将得到的消化肽从凝胶释放到溶液中并用于肽质量分析。

    Selective labeling agent for phosphoproteome analysis and phosphorylated site analysis
    2.
    发明授权
    Selective labeling agent for phosphoproteome analysis and phosphorylated site analysis 失效
    磷酸化蛋白分析和磷酸化位点分析的选择性标记试剂

    公开(公告)号：US07250266B2

    公开(公告)日：2007-07-31

    申请号：US10907945

    申请日：2005-04-21

    申请人： Yeong Hee Ahn ,  Jong Shin Yoo ,  Jin Young Kim ,  Kun Cho

    发明人： Yeong Hee Ahn ,  Jong Shin Yoo ,  Jin Young Kim ,  Kun Cho

    IPC分类号： G01N33/00

    CPC分类号： G01N33/6848 ,  A61K31/155 ,  G01N33/6803 ,  G01N33/6842 ,  G01N2458/15

    摘要： Disclosed is a method of analyzing mass of the phosphoproteins or phosphopeptides and of analyzing phosphorylated positions at a phosphoprotein or phosphopeptide, comprising the steps of: 1) dephosphorylating at least one Ser and/or Thr residue of the phosphoprotein or phosphopeptide; 2) tagging the dephosphorylated amino acid residues with a tag having a R-L-G moiety wherein R is a nucleophilic functional group that selectively bind with dephosphorylated amino acid residues, G is selected from the group consisting of guanidine moiety or protected guanidine moiety such as a mono-N-protected guanidino group, a di-N,N′-protected guanidino group and an N′-protected guanidino group, and L is a linker linking the R and the G; and 3) subjecting the tagged proteins or peptides to mass spectrometry. The method is capable of precisely analyzing mass of phosphoproteins of trace amounts as well as positions of phosphoryated amino acids.

    摘要翻译： 公开了一种分析磷蛋白或磷酸肽的质量并分析磷蛋白或磷酸肽的磷酸化位置的方法，包括以下步骤：1）使磷酸蛋白或磷酸肽的至少一个Ser和/或Thr残基去磷酸化; 2）用具有RLG部分的标签标记去磷酸化的氨基酸残基，其中R是与去磷酸化的氨基酸残基选择性结合的亲核官能团，G选自胍部分或被保护的胍部分，例如单 - N-保护的胍基，二-N，N'保护的胍基和N'保护的胍基，L是连接R和G的连接基团; 和3）对标记的蛋白质或肽进行质谱分析。 该方法能够精确分析痕量的磷蛋白质量以及磷酸化氨基酸的位置。

    In-Gel Tagging And In-Gel Digestion For Phosphoproteins Analysis And Phosphorylation Site Identification
    3.
    发明申请
    In-Gel Tagging And In-Gel Digestion For Phosphoproteins Analysis And Phosphorylation Site Identification 失效
    凝胶标记和凝胶消化磷蛋白分析和磷酸化位点鉴定

    公开(公告)号：US20070287169A1

    公开(公告)日：2007-12-13

    申请号：US11574660

    申请日：2004-10-08

    申请人： Yeong Ahn ,  Jong Yoo ,  Jae Lee ,  Jin Kim ,  Kun Cho

    发明人： Yeong Ahn ,  Jong Yoo ,  Jae Lee ,  Jin Kim ,  Kun Cho

    IPC分类号： C12P21/06 ,  C07K14/00

    CPC分类号： G01N33/6842 ,  G01N33/6848

    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.

    摘要翻译： 本发明涉及用位点特异性标记试剂对磷酸化蛋白质进行磷酸化位点特异性标记的方法，并分析得到的标记物，更具体地说，一种用于原位标记保留在聚合物凝胶中的磷酸化蛋白的磷酸化位点的方法， 亲核标记试剂。 它还涉及通过适当选择亲核标记试剂在以前的磷酸化位点产生新的蛋白水解可切割位点的方法。 它还涉及通过凝胶内消化先前凝胶内标记蛋白的磷酸肽分析和磷酸化位点鉴定的方法以及随后对所得肽的质量分析。 本发明提供了凝胶化学标记方法，用于保留在聚合物凝胶基质中的磷酸蛋白的磷酸氨基酸残基。 磷酸蛋白可以通过各种方法如凝胶电泳固定在凝胶基质中。 在磷酸化蛋白质的磷酸化氨基酸残基的标签反应期间，将固定的磷酸蛋白质保留在凝胶基质中，并且所得到的标记蛋白质也保留在凝胶基质中，直到完成标记试剂的洗涤之后的纯化步骤。 标记的蛋白质被蛋白酶消化，并将得到的消化肽从凝胶释放到溶液中并用于肽质量分析。