Proteomic screening method for phosphopeptide motif binding proteins using peptide libraries.
Proteomic screening method for phosphopeptide motif binding proteins using peptide libraries.
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DOI：
10.1021/pr200578n
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发表时间：
2011-09-02
影响因子：
4.4
通讯作者：
Asara, John M.
中科院分区：
文献类型：
--
作者：
Christofk, Heather R.;Wu, Ning;Cantley, Lewis C.;Asara, John M.
关键词：
磷酸肽结合结构域介导细胞内激酶信号传导所必需的蛋白质复合物的定向和局部组装。为了鉴定磷酸肽结合蛋白，我们开发了一种使用固定化部分简并磷酸肽混合物结合SILAC和微毛细管LC/MS/MS的蛋白质组学筛选方法。该方法用于鉴定与磷酸化肽库亲和基质特异性结合的蛋白质，包括pTyr和基序pSer/pThr-Pro，pSer/pThr-X-X-X-pSer/pThr，pSer/pThr-Glu/Asp或pSer/pThr-pSer/pThr。使重和轻SILAC裂解物分别流过含有这些磷酸化和非磷酸化（对照）肽文库的柱，并洗脱、合并、消化结合的蛋白质，并使用混合四极杆-TOF质谱仪通过LC/MS/MS进行分析。计算重：轻肽离子比，并且认为产生比大于约3：1的肽来自潜在的磷酸肽结合蛋白，因为该比代表已知阳性对照的最低比。其中许多是已知的磷酸肽结合蛋白，包括与pTyr结合的含有PI 3 K的p85亚基的SH 2结构域、与pSer/pThr-Asp/Glu结合的14-3-3、与pSer/pThr-Pro结合的含有PLK 1和Pin 1的polo-box结构域以及与pTyr结合的丙酮酸激酶M2。通过肽库筛选鉴定的命中中约有一半是新的。蛋白质结构域富集分析显示，大多数pTyr命中物含有SH 2结构域，如预期的，并且在较小程度上含有SH 3、C1、STAT、Tyr磷酸酶、P激酶、C2和PH结构域，然而，pSer/pThr基序没有显示在命中物之间富集的结构域。
Phosphopeptide binding domains mediate the directed and localized assembly of protein complexes essential to intracellular kinase signaling. To identify phosphopeptide binding proteins, we developed a proteomic screening method using immobilized partially-degenerate phosphopeptide mixtures combined with SILAC and microcapillary LC/MS/MS. The method was used to identify proteins that specifically bound to phosphorylated peptide library affinity matrices, including pTyr, and the motifs pSer/pThr-Pro, pSer/pThr-X-X-X-pSer/pThr, pSer/pThr-Glu/Asp or pSer/pThr-pSer/pThr in degenerate sequence contexts. Heavy and light SILAC lysates were flowed over columns containing these phosphorylated and non-phosphorylated (control) peptide libraries respectively, and bound proteins were eluted, combined, digested and analyzed by LC/MS/MS using a hybrid quadrupole-TOF mass spectrometer. Heavy:light peptide ion ratios were calculated, and peptides that yielded ratios greater than ~3:1 were considered as being from potential phosphopeptide binding proteins since this ratio represents the lowest ratio from a known positive control. Many of those identified were known phosphopeptide-binding proteins, including the SH2 domain containing p85 subunit of PI3K bound to pTyr, 14-3-3 bound to pSer/pThr-Asp/Glu, polo-box domain containing PLK1 and Pin1 bound to pSer/pThr-Pro and pyruvate kinase M2 binding to pTyr. Approximately half of the hits identified by the peptide library screens were novel. Protein domain enrichment analysis revealed that most pTyr hits contain SH2 domains, as expected and to lesser extent SH3, C1, STAT, Tyr phosphatase, Pkinase, C2 and PH domains, however, pSer/pThr motifs did not reveal enriched domains across hits.
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影响因子：
64.5
作者：
Huttlin EL;Jedrychowski MP;Elias JE;Goswami T;Rad R;Beausoleil SA;Villén J;Haas W;Sowa ME;Gygi SP
通讯作者：
Gygi SP
影响因子：
9.9
作者：
Lange, Vinzenz;Picotti, Paola;Domon, Bruno;Aebersold, Ruedi
通讯作者：
Aebersold, Ruedi
影响因子：
3.4
作者：
Asara, John M.;Christofk, Heather R.;Cantley, Lewis C.
通讯作者：
Cantley, Lewis C.
影响因子：
4.8
作者：
Kirkpatrick, DS;Gerber, SA;Gygi, SP
通讯作者：
Gygi, SP
影响因子：
7
作者：
Ross, PL;Huang, YLN;Pappin, DJ
通讯作者：
Pappin, DJ