Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry.

Determination of Phosphohistidine Stoichiometry in Histidine Kinases by Intact Mass Spectrometry.
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通过完整质谱法测定组氨酸激酶中的磷酸组氨酸化学计量。

DOI:
10.1007/978-1-4939-9884-5_6
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发表时间:
2020
期刊:
Methods in molecular biology (Clifton, N.J.)
影响因子:
--
通讯作者:
Tomlinson LJ
Tomlinson LJ
中科院分区:
--
文献类型:
--
作者:
Tomlinson LJ

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由于分析相对不稳定的含磷酸组氨酸的蛋白质的挑战,蛋白质组氨酸磷酸化在很大程度上仍然未被探索。我们描述了一个程序,用于确定化学计量的组氨酸磷酸化的人组氨酸激酶NME1和NME2的完整质谱保留这种酸不稳定的蛋白质修饰的条件下。通过在不存在和存在合适的磷酸供体的情况下表征这两种模型组氨酸蛋白激酶,可以确定组氨酸磷酸化的化学计量。所描述的方法可以很容易地适用于分析其他蛋白质含有磷酸组氨酸。
Protein histidine phosphorylation has largely remained unexplored due to the challenges of analyzing relatively unstable phosphohistidine-containing proteins. We describe a procedure for determining the stoichiometry of histidine phosphorylation on the human histidine kinases NME1 and NME2 by intact mass spectrometry under conditions that retain this acid-labile protein modification. By characterizing these two model histidine protein kinases in the absence and presence of a suitable phosphate donor, the stoichiometry of histidine phosphorylation can be determined. The described method can be readily adapted for the analysis of other proteins containing phosphohistidine.
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影响因子: 4
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影响因子: 7.4
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