Rigorous determination of the stoichiometry of protein phosphorylation using mass spectrometry.
Quantification of the stoichiometry of phosphorylation is usually achieved using a mixture of phosphatase treatment and differential isotopic labeling. Here, we introduce a new approach to the concomitant determination of absolute protein concentration and the stoichiometry of phosphorylation at predefined sites. The method exploits QconCAT to quantify levels of phosphorylated and nonphosphorylated peptide sequences in a phosphoprotein. The nonphosphorylated sequence is used to determine the absolute protein quantity and serves as a reference to calculate the extent of phosphorylation at the second peptide. Thus, the stoichiometry of phosphorylation and the absolute protein concentration can be determined accurately in a single experiment.
Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, School of Chemistry, University of Manchester, Manchester, United Kingdom.
Hannah
H
Johnson
Claire E
CE
Eyers
Patrick A
PA
Eyers
Robert J
RJ
Beynon
Simon J
SJ
Gaskell
D000465:Algorithms; D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010748:Phosphopeptides / Q000032:analysis / Q000737:chemistry*; D010766:Phosphorylation; D020539:Sequence Analysis, Protein / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010748:Phosphopeptides