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Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae
Published on: October 13, 2009
Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide
M B Goshe1, T P Conrads, E A Panisko
1Environmental and Molecular Sciences Laboratory, Richland, Washington 99352, USA.
Analytical Chemistry
|June 14, 2001
Summary
A novel phosphoprotein isotope-coded affinity tag (PhIAT) method enables quantitative analysis of protein O-phosphorylation. This technique combines stable isotope and biotin labeling for precise O-phosphorylation state measurements.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- O-phosphorylation is a crucial post-translational modification regulating protein function.
- Accurate quantitative measurement of O-phosphorylation states is essential for understanding cellular processes.
- Existing methods for O-phosphorylation analysis can be complex and time-consuming.
Purpose of the Study:
- To develop a novel method for the enrichment and quantitative measurement of protein O-phosphorylation states.
- To introduce the phosphoprotein isotope-coded affinity tag (PhIAT) labeling approach.
- To demonstrate the utility of PhIAT for analyzing O-phosphorylation in complex biological samples.
Main Methods:
- Development of the PhIAT reagent, integrating stable isotope labeling and biotinylation.
- Hydroxide ion-mediated beta-elimination of O-phosphate followed by addition of deuterated or non-deuterated ethanedithiol.
- Biotinylation of the modified moiety and subsequent affinity enrichment using immobilized avidin.
- Analysis of labeled peptides by capillary reversed-phase liquid chromatography-mass spectrometry.
Main Results:
- Successful synthesis of the PhIAT reagent, simplifying O-phosphorylation derivatization to a one-step process.
- Demonstration of PhIAT using the model phosphoprotein beta-casein, with isolation and identification of labeled phosphopeptides.
- Application of the PhIAT method to a yeast protein extract, showing its applicability to complex proteomes.
- Reliable quantitative measurements of differences in protein O-phosphorylation states were achieved.
Conclusions:
- The PhIAT labeling technique offers a robust and efficient approach for quantitative O-phosphorylation analysis.
- This method facilitates the study of dynamic changes in protein phosphorylation.
- PhIAT has significant potential for advancing phosphoproteomics research.
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