The Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre (MIB), Manchester, United Kingdom.
Anal Chem. 2012 Sep 4;84(17):7384-92. doi: 10.1021/ac301038u. Epub 2012 Aug 17.
Reversible phosphorylation regulates the majority of intracellular networking and pathways. The study of this widely explored post-translational modification is usually challenged by low stoichiometric levels of modification. Many approaches have been developed to overcome this problem and to achieve rigorous characterization of protein phosphorylation. We describe a method for enhanced detection of low-abundance protein phosphorylation that uses selective introduction of (18)O label into phosphorylation sites with H(2)(18)O and mass spectrometric detection. The method was applied to introduce (18)O label into bacterially expressed Aurora A kinase phosphorylation sites and resulted in the representation of phosphorylated peptides as doublets or triplets according to the number of phosphate groups. A total of 28 phosphopeptides were observed by this method.
可逆磷酸化调节着大多数的细胞内网络和途径。对这种广泛研究的翻译后修饰的研究通常受到修饰的低化学计量水平的挑战。已经开发了许多方法来克服这个问题，并实现对蛋白质磷酸化的严格表征。我们描述了一种用于增强检测低丰度蛋白质磷酸化的方法，该方法使用 H(2)(18)O 将(18)O 标记选择性地引入磷酸化位点，并通过质谱检测。该方法被应用于将(18)O 标记引入细菌表达的 Aurora A 激酶磷酸化位点，结果根据磷酸基团的数量，磷酸化肽以双峰或三峰的形式出现。通过这种方法观察到了总共 28 个磷酸肽。
