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Physiol. Genomics (August 7, 2007). doi:10.1152/physiolgenomics.00096.2007
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Submitted on April 27, 2007
Accepted on August 5, 2007

Relative quantification of peptide phosphorylation in a complex mixture using 18O labeling

Julia Smith1, Michael Olivier2, and Andrew S Greene3*

1 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
2 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States; Human & Molecular Genetics Center, Dept. of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States
3 Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, United States; Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin, United States

* To whom correspondence should be addressed. E-mail: agreene{at}mcw.edu.

We have developed a method to determine the degree of phosphorylation of a peptide in a complex mixture without enrichment or operation of the mass spectrometer in negative ion mode. Yeast lysate containing known amounts of synthetic peptides (VPQLEIVPNSAEERLHSMK and VPQLEIVPN[pS]AEERLHSMK) was labeled with 16O and 18O during hydrolysis. After treatment of one sample with a cocktail of phosphatases the two samples were pooled. The intensity of the dephosphorylated peptide peaks was used to infer the degree of phosphorylation present before treatment. The linear dynamic range of this method is greater than ten fold before either of the peptide envelopes becomes indistinguishable from the surrounding noise. Since both the site of post translational modification, and the proportion of the protein population that is modified is vital in protein function, the employment of this technique will provide a valuable tool for the analysis of the functional implications of protein phosphorylation.




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S. P. Mirza and M. Olivier
Methods and approaches for the comprehensive characterization and quantification of cellular proteomes using mass spectrometry
Physiol Genomics, March 10, 2008; 33(1): 3 - 11.
[Abstract] [Full Text] [PDF]


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