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Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.

ABSTRACT: 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) with stable-isotope-labeled standard peptides. In contrast with classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only one nonphosphorylated SIS (stable isotope-coded standard) and two analyses (one for the untreated sample and one for the phosphatase-treated sample), from which the expression and modification levels can accurately be determined. From these analyses, the percent 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 (ERalpha and HER2), and for phosphorylated RAF and ERK1, which also contain phosphorylation sites of biological importance. 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 ERalpha protein revealed that the PPQ-MRM method 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.

SUBMITTER: Domanski D

PROVIDER: S-EPMC2909760 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.

Domanski Dominik D Murphy Leigh C LC Borchers Christoph H CH

Analytical chemistry 20100701 13

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) with stable-isotope-labeled standard peptides. In contrast with classical MRM methods for the quantitation of phosphorylation stoichiometry, the PPQ-MRM method needs only ...[more]

PMID: 20524616

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Project description:Protein phosphorylation, one of the most common and important modifications of acute and reversible regulation of protein function, plays a dominant role in almost all cellular processes. These signaling events regulate cellular responses, including proliferation, differentiation, metabolism, survival, and apoptosis. Several studies have been successfully used to identify phosphorylated proteins and dynamic changes in phosphorylation status after stimulation. Nevertheless, it is still rather difficult to elucidate precise complex phosphorylation signaling pathways. In particular, how signal transduction pathways directly communicate from the outer cell surface through cytoplasmic space and then directly into chromatin networks to change the transcriptional and epigenetic landscape remains poorly understood. Here, we describe the optimization and comparison of methods based on thiophosphorylation affinity enrichment, which can be utilized to monitor phosphorylation signaling into chromatin by isolation of phosphoprotein containing nucleosomes, a method we term phosphorylation-specific chromatin affinity purification (PS-ChAP). We utilized this PS-ChAP(1) approach in combination with quantitative proteomics to identify changes in the phosphorylation status of chromatin-bound proteins on nucleosomes following perturbation of transcriptional processes. We also demonstrate that this method can be employed to map phosphoprotein signaling into chromatin containing nucleosomes through identifying the genes those phosphorylated proteins are found on via thiophosphate PS-ChAP-qPCR. Thus, our results showed that PS-ChAP offers a new strategy for studying cellular signaling and chromatin biology, allowing us to directly and comprehensively investigate phosphorylation signaling into chromatin to investigate if these pathways are involved in altering gene expression. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD002436.

Dataset Information

Assay development for the determination of phosphorylation stoichiometry using multiple reaction monitoring methods with and without phosphatase treatment: application to breast cancer signaling pathways.

Publications

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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