Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay.
Despite recent advancements in large-scale phosphoproteomics, methods to quantify kinase-specific phosphorylation stoichiometry of protein substrates are lacking. We developed a method to quantify kinase-specific phosphorylation stoichiometry by combining the reverse in-gel kinase assay (RIKA) with high-resolution liquid chromatography-mass spectrometry (LC-MS). Beginning with predetermined ratios of phosphorylated to nonphosphorylated protein kinase CK2 (CK2) substrate molecules, we employed18O-labeled adenosine triphosphate (18O-ATP) as the phosphate donor in a RIKA, then quantified the ratio of18O- versus16O-labeled tryptic phosphopeptide using high mass accuracy mass spectrometry (MS). We demonstrate that the phosphorylation stoichiometry determined by this method across a broad percent phosphorylation range correlated extremely well with the predicted value (correlation coefficient = 0.99). This approach provides a quantitative alternative to antibody-based methods of determining the extent of phosphorylation of a substrate pool.
Department of Biological Sciences, University of Maryland Baltimore County , Baltimore, Maryland 21250, United States.
Xiang
X
Li
Biological Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
Jonathan T
JT
Cox
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland , Baltimore, Maryland 21201, United States.
Weiliang
W
Huang
Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland , Baltimore, Maryland 21201, United States.
Maureen
M
Kane
Biological Sciences Division, Pacific Northwest National Laboratory , Richland, Washington 99352, United States.
Keqi
K
Tang
Department of Biological Sciences, University of Maryland Baltimore County , Baltimore, Maryland 21250, United States.|Marlene and Stewart Greenebaum Cancer Center, University of Maryland Baltimore , Baltimore, Maryland 21201, United States.
Charles J
CJ
Bieberich
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D047390:Casein Kinase II / Q000737:chemistry* / Q000378:metabolism; D005782:Gels / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling; D010103:Oxygen Isotopes; D010748:Phosphopeptides / Q000032:analysis* / Q000378:metabolism; D010766:Phosphorylation
D005782:Gels; D010103:Oxygen Isotopes; D010748:Phosphopeptides; D000255:Adenosine Triphosphate; D047390:Casein Kinase II