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Sensitive measurement of total protein phosphorylation level in complex protein samples.

ABSTRACT: Measurement of protein phosphorylation plays an essential role in delineating cell signaling pathways. Although the detection of a specific phosphoprotein has been largely accomplished by immunological methods, a specific and sensitive assay to measure total protein phosphorylation level in complex samples such as whole cell extracts has yet to be established. Here, we present a sensitive phosphorylation assay on a microwell plate to determine total protein phosphorylation level calibrated to a phosphoprotein standard. The core of the assay is a reagent termed pIMAGO that is multi-functionalized with titanium ions for its superior selectivity towards phosphorylated proteins and with fluorophores for quantification. The specificity, sensitivity, and quantitative nature of the assay were demonstrated with standard proteins and whole cell lysates. The method was then employed to measure the overall protein phosphorylation level of human cells under different treatments. At last, we investigated the practicability of the assay to serve as a sensitive tool to estimate the amount of phosphorylated samples prior to a mass spectrometry-based phosphoproteomic analysis.

SUBMITTER: Pan L

PROVIDER: S-EPMC4425989 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Sensitive measurement of total protein phosphorylation level in complex protein samples.

Pan Li L Wang Linna L Hsu Chuan-Chih CC Zhang Jiazhen J Iliuk Anton A Tao W Andy WA

The Analyst 20150410 10

Measurement of protein phosphorylation plays an essential role in delineating cell signaling pathways. Although the detection of a specific phosphoprotein has been largely accomplished by immunological methods, a specific and sensitive assay to measure total protein phosphorylation level in complex samples such as whole cell extracts has yet to be established. Here, we present a sensitive phosphorylation assay on a microwell plate to determine total protein phosphorylation level calibrated to a ...[more]

PMID: 25857711

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Project description:It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on (32)P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria.

Dataset Information

Sensitive measurement of total protein phosphorylation level in complex protein samples.

Publications

Sensitive measurement of total protein phosphorylation level in complex protein samples.

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