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.

[1]  M. Mann,et al.  Quantitative Phosphoproteomics Applied to the Yeast Pheromone Signaling Pathway*S , 2005, Molecular & Cellular Proteomics.

[2]  P. Eyers,et al.  Phosphoregulation of human Mps1 kinase. , 2009, The Biochemical journal.

[3]  D. Matthews,et al.  Quantification of Protein Phosphorylation by Liquid Chromatography−Mass Spectrometry , 2008, Analytical chemistry.

[4]  K. Resing,et al.  Mps1 Activation Loop Autophosphorylation Enhances Kinase Activity* , 2007, Journal of Biological Chemistry.

[5]  M. Kirschner,et al.  Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[6]  M. Sussman,et al.  An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins , 2004, Journal of the American Society for Mass Spectrometry.

[7]  Natalie G. Ahn,et al.  Identification of Novel Phosphorylation Sites on Xenopus laevis Aurora A and Analysis of Phosphopeptide Enrichment by Immobilized Metal-affinity Chromatography * , 2003, Molecular & Cellular Proteomics.

[8]  R. Beynon,et al.  Multiplexed absolute quantification in proteomics using artificial QCAT proteins of concatenated signature peptides , 2005, Nature Methods.

[9]  S. Carr,et al.  N-Terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry. , 2002, Rapid communications in mass spectrometry : RCM.

[10]  S. Hanks,et al.  Assaying protein kinase activity. , 2004, Methods in molecular biology.

[11]  M. Mann,et al.  Global, In Vivo, and Site-Specific Phosphorylation Dynamics in Signaling Networks , 2006, Cell.

[12]  Hanno Steen,et al.  Phosphorylation Analysis by Mass Spectrometry , 2006, Molecular & Cellular Proteomics.

[13]  Robert J Beynon,et al.  QCAL—a novel standard for assessing instrument conditions for proteome analysis , 2008, Journal of the American Society for Mass Spectrometry.

[14]  Linfeng Wu,et al.  Absolute Quantification of Multisite Phosphorylation by Selective Reaction Monitoring Mass Spectrometry , 2006, Molecular & Cellular Proteomics.

[15]  Leonard M. G. Chavas,et al.  Crystal Structure of the Catalytic Domain of the Mitotic Checkpoint Kinase Mps1 in Complex with SP600125* , 2008, Journal of Biological Chemistry.

[16]  S. Hanks,et al.  Absolute quantification of phosphorylation on the kinase activation loop of cellular focal adhesion kinase by stable isotope dilution liquid chromatography/mass spectrometry. , 2009, Analytical chemistry.

[17]  Hanno Steen,et al.  FLEXIQuant: a novel tool for the absolute quantification of proteins, and the simultaneous identification and quantification of potentially modified peptides. , 2009, Journal of proteome research.

[18]  R. Beynon,et al.  Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes , 2006, Nature Protocols.

[19]  E. Krause,et al.  The relative influence of phosphorylation and methylation on responsiveness of peptides to MALDI and ESI mass spectrometry. , 2009, Journal of mass spectrometry : JMS.

[20]  T. Pawson,et al.  Reading protein modifications with interaction domains , 2006, Nature Reviews Molecular Cell Biology.

[21]  P. Cohen,et al.  Identification of a phosphorylation site on skeletal muscle myosin light chain kinase that becomes phosphorylated during muscle contraction. , 2002, Archives of biochemistry and biophysics.

[22]  S. Gygi,et al.  Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[23]  C. Ruse,et al.  Quantitative dynamics of site-specific protein phosphorylation determined using liquid chromatography electrospray ionization mass spectrometry. , 2002, Analytical chemistry.