Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS 1)
Phosphorylation sites of Arabidopsis MAP kinase substrate 1 (MKS 1)
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DOI：
10.1016/j.bbapap.2007.07.002
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发表时间：
2007-09-01
影响因子：
3.2
通讯作者：
Svensson, Birte
中科院分区：
文献类型：
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作者：
Caspersen, Mikael B.;Qiu, Jin-Long;Svensson, Birte
拟南芥MAP激酶4（MPK4）底物MKS 1在大肠杆菌中表达，纯化的全长，6x组氨酸（His）标记的MKS 1在体外磷酸化的血凝素（HA）标记的MPK 4免疫沉淀从植物。最初通过电泳和ProQ Diamond凝胶染色验证MKS 1磷酸化，并通过胰蛋白酶或胰凝乳蛋白酶消化蛋白质以获得最大序列覆盖率，以便于鉴定磷酸化位置。在通过质谱法分析之前，将样品脱盐、通过TiO 2或两者以改进磷酸肽检测。由于MAP激酶通常磷酸化丝氨酸或苏氨酸，然后磷酸化脯氨酸（Ser/Thr-Pro），因此计算潜在磷酸化肽的理论质量，并将与这些质量匹配的质谱峰片段化，并搜索指示磷酸化的类似于98 Da的中性丢失信号。此外，MPK 4处理的MKS 1中存在的质谱峰（但未处理的MKS 1的对照肽图谱中不存在）被片段化。对片段化光谱进行MASCOT数据库检索，其鉴定了磷酸化形式的12个Ser-Pro丝氨酸残基中的3个（Ser72、Set108、Ser120）。(c)2007 Elsevier B.V.保留所有权利。
The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophoresis and gel-staining with ProQ Diamond and the protein was digested by either trypsin or chymotrypsin for maximum sequence coverage to facilitate identification of phosphorylated positions. Prior to analysis by mass spectrometry, samples were either desalted, passed over TiO2 or both for improved phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal at similar to 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine residues (Ser72, Set108, Ser120) in the phosphorylated form. (c) 2007 Elsevier B.V. All rights reserved.