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Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry
- Source :
- Analytical chemistry. 74(7)
- Publication Year :
- 2002
-
Abstract
- We have developed and validated a method that uses liquid chromatography/electrospray ionization-mass spectrometry to quantify site-specific protein phosphorylation. The method uses selected ion monitoring to determine the chromatographic peak areas of specific tryptic peptides from the protein of interest. The extent of phosphorylation is determined from the ratio of the phosphopeptide peak area to the peak area of an unmodified reference peptide that acts as internal standard, correcting for variations in protein amounts and peptide recovery in the digest preparation procedure. As a result, we refer to this protocol as the native reference peptide method. Mole of phosphate at the selected site per mole of protein is obtained from this ratio, using calibration curves of synthetic peptides to determine relative responses. Our method begins with protein separation by SDS-PAGE and is carried out on amounts of peptide produced by an in-gel digestion of single Coomassie blue-stained bands. To illustrate the utility of the method and provide validation, we used cardiac troponin I as analyte and monitored the time course of a protein kinase C betaII reaction. Those analyses appropriately demonstrate the time-dependent increase of phosphorylation at a PKC-preferred site, Ser44 in the peptide 41ISASPR45 and the concomitant consumption of the nonphosphorylated peptide. We believe that this method provides a novel tool to directly measure specific phosphorylation sites in proteins in different physiological states and expect that the method will be adaptable not only to a variety of samples types (i.e., culture cells, tissues, etc.) but to a variety of posttranslation modifications as well.
- Subjects :
- chemistry.chemical_classification
Spectrometry, Mass, Electrospray Ionization
Chromatography
Binding Sites
Protein mass spectrometry
Electrospray ionization
Troponin I
Analytical chemistry
Peptide
Mass spectrometry
Phosphoproteins
Sample preparation in mass spectrometry
Analytical Chemistry
Phosphates
Kinetics
chemistry
Peptide mass fingerprinting
Protein purification
Protein Kinase C beta
Animals
Humans
Selected ion monitoring
Phosphorylation
Peptides
Protein Kinase C
Subjects
Details
- ISSN :
- 00032700
- Volume :
- 74
- Issue :
- 7
- Database :
- OpenAIRE
- Journal :
- Analytical chemistry
- Accession number :
- edsair.doi.dedup.....3d915916b5b301e52763712a66b34a37