Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.
Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.
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DOI：
10.1021/ac1005553
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发表时间：
2010-07-01
影响因子：
7.4
通讯作者：
Borchers, Christoph H.
中科院分区：
文献类型：
--
作者：
Domanski, Dominik;Murphy, Leigh C.;Borchers, Christoph H.
我们开发了一种基于磷酸酶的磷酸肽定量（PPQ）方法，用于确定复杂生物样品中的磷酸化化学计量。该PPQ方法基于酶促去磷酸化，结合使用稳定同位素标记的标准肽通过多反应监测（MRM）检测进行的特异性和准确的肽鉴定和定量。与用于磷酸化化学计量定量的经典MRM方法相比，PPQ-MRM方法仅需要一个非磷酸化SIS（稳定同位素编码的标准品）和两个分析（一个用于未处理的样品，一个用于磷酸酶处理的样品），由此可以准确地确定表达和修饰水平。从这些分析中，可以确定磷酸化%。在本文中，我们比较了PPQ-MRM方法与不含磷酸酶的MRM方法，并证明了这些方法在检测和定量经典磷酸化乳腺癌生物标志物（ERα和HER 2）以及磷酸化RAF和ERK 1磷酸化中的应用，这些磷酸化RAF和ERK 1也包含具有重要生物学意义的磷酸化位点。使用掺入复杂蛋白质消化物中的合成肽，我们能够使用我们的PPQ-MRM方法准确测定特定肽的总磷酸化化学计量，以及存在的肽和磷酸肽的绝对量。对含有ERα蛋白的样品的分析表明，PPQ-MRM能够测定细胞系蛋白质中的磷酸化化学计量，并且在磷酸化和总蛋白量方面与使用直接MRM方法获得的测定结果具有良好的一致性。
We have developed a phosphatase-based phosphopeptide quantitation (PPQ) method for determining phosphorylation stoichiometry in complex biological samples. This PPQ method is based on enzymatic dephosphorylation, combined with specific and accurate peptide identification and quantification by multiple reaction monitoring (MRM) detection with stable-isotope-labeled standard peptides. In contrast with the classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one non-phosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the % phosphorylation can be determined. In this manuscript, we compare the PPQ-MRM method with an MRM method without phosphatase, and demonstrate the application of these methods to the detection and quantitation of phosphorylation of the classic phosphorylated breast cancer biomarkers (ERα and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites with important biological implications. Using synthetic peptides spiked into a complex protein digest, we were able to use our PPQ-MRM method to accurately determine the total phosphorylation stoichiometry on specific peptides, as well as the absolute amount of the peptide and phosphopeptide present. Analyses of samples containing ERα protein revealed that the PPQ-MRM is capable of determining phosphorylation stoichiometry in proteins from cell lines, and is in good agreement with determinations obtained using the direct MRM approach in terms of phosphorylation and total protein amount.
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影响因子：
--
作者：
ARNOLD, SF;OBOURN, JD;NOTIDES, AC
通讯作者：
NOTIDES, AC
影响因子：
7
作者：
Kuzyk, Michael A.;Smith, Derek;Borchers, Christoph H.
通讯作者：
Borchers, Christoph H.
影响因子：
46.9
作者：
Ficarro, SB;McCleland, ML;White, FM
通讯作者：
White, FM
DOI：
10.1016/j.jasms.2008.02.008
发表时间：
2008-05-01
影响因子：
3.2
作者：
Britton, David J.;Scott, Gary K.;Baldwin, Michael A.
通讯作者：
Baldwin, Michael A.
DOI：
10.2174/1874189400802010018
发表时间：
2008-05-27
期刊：
The open clinical cancer journal
影响因子：
--
作者：
Guo, Lan;Abraham, Jame;Qian, Yong
通讯作者：
Qian, Yong