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Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling.
Julia R Smith1, Michael Olivier, Andrew S Greene
1Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Physiological Genomics
|August 9, 2007
Summary
This study introduces a new method to quantify peptide phosphorylation in complex mixtures. The technique uses oxygen isotope labeling and mass spectrometry to determine phosphorylation levels without specialized equipment.
Area of Science:
- Biochemistry
- Proteomics
- Analytical Chemistry
Background:
- Protein phosphorylation is a critical post-translational modification regulating cellular processes.
- Accurate quantification of phosphorylation is essential for understanding protein function.
- Existing methods often require enrichment or specific mass spectrometer settings.
Purpose of the Study:
- To develop a novel, simplified method for determining the degree of peptide phosphorylation.
- To enable quantification in complex biological mixtures without specialized mass spectrometry conditions.
- To provide a tool for analyzing the functional implications of protein phosphorylation.
Main Methods:
- Utilized oxygen-16 and oxygen-18 isotope labeling during peptide hydrolysis.
- Employed a phosphatase treatment on one sample followed by pooling with an untreated sample.
- Quantified dephosphorylated peptide peak intensities to infer original phosphorylation levels.
- Operated mass spectrometer in positive ion mode, avoiding negative ion mode requirements.
Main Results:
- Successfully determined the degree of phosphorylation in a complex yeast lysate mixture.
- Demonstrated a linear dynamic range of over 10-fold for the method.
- The method is robust and does not require peptide enrichment steps.
- Avoided the need for negative ion mode operation on the mass spectrometer.
Conclusions:
- The developed method offers a valuable tool for analyzing protein phosphorylation.
- It simplifies the quantification of phosphorylation in complex biological samples.
- This technique facilitates the study of functional implications of post-translational modifications.
