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, 88 (23), 11468-11475

Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay


Quantifying Kinase-Specific Phosphorylation Stoichiometry Using Stable Isotope Labeling In a Reverse In-Gel Kinase Assay

Xiang Li et al. Anal Chem.


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 employed 18O-labeled adenosine triphosphate (18O-ATP) as the phosphate donor in a RIKA, then quantified the ratio of 18O- versus 16O-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.


Figure 1
Figure 1. Measurement of kinase-specific phosphorylation stoichiometry
Circles represent protein molecules that are substrates of a given kinase. Top left: Of nine substrate molecules six are shown as phosphorylated (red,16P). After phosphorylation to completion in a RIKA, the remaining three substrate molecules become labeled with 18O-phosphate (Top right, 18P). Phosphopeptides enriched after tryptic digestion are analyzed by LC-MS. The ratio of 16O-phosphorylated peptide to 18O-phosphorylated peptide is 6:3, from which the absolute phosphorylation stoichiometry is calculated to be: [6/(6+3)]*100%=66.7%. Bottom left: When the kinase activity is partially inhibited, two more substrate molecules in the pool of nine are not phosphorylated. In this case, the measured 16O:18O ratio would be 4:5; and phosphorylation stoichiometry = 44.4%.
Figure 2
Figure 2. Measuring 18O:16O phosphorylation ratio by LC-MS
Representative LC-MS spectrum measuring 16O:18O phosphorylation ratio for peptide 108–122 DWEDDSDE (Sp) DEDMSNFDR from TEBP. This ion is 3+ charged, with a m/z value of 652.55 for the 16O-phosphorylated form, and a m/z value of 654.55 for the 18O-phosphorylated form. A. Spectrum for a premixed 8:2 ratio. The measured ratio is 100:21; and the expected value is 100:25. B. Spectrum for 2:8 ratio. The measured value is 31.5:100, and the expected value is 25:100. Arrows, phosphorylated ion peaks. Relative abundance values are indicated in parentheses.
Figure 3
Figure 3. CK2 substrates are quantitatively phosphorylated in RIKA
A. TEBP is phosphorylated close to completion in RIKA. ~10 μg recombinant TEBP was analyzed in a CK2 RIKA (20 μg/ml in gel, 50 μM 16O-ATP in reaction buffer) (left panel). A control gel without ATP was analyzed in parallel (not shown). To determine the extent of TEBP phosphorylation, the Coomassie blue-stained TEBP band was excised and homogenized. TEBP was extracted and analyzed in a second RIKA (right panel) with 32P-ATP as the phosphate donor. A 10-fold dilution of extracted TEBP was included to facilitate evaluation of phosphorylation efficiency. Lanes 1&2, 3&4, 5&6, and 7&8 are technical replicates. B&C. Measuring the efficiency of phosphorylation of TEBP in RIKA by using label-free LC-MS. B. The spectrum and intensities of the precursor ions for the reference peptide (gray), and for the non-phosphorylated peptide (blue) in the control gel. Raw spectra were de-convoluted and de-isotoped as described in the experimental section. C. The intensities of the precursor ions for the reference peptide (gray), the non-phosphorylated peptide (blue), and the phosphorylated peptide in the experimental gel.
Figure 4
Figure 4. CK2-specific phosphorylation stoichiometry can be accurately quantified
Stoichiometry values obtained by quantitative MS were plotted against expected values (after adjustment for 18O-ATP purity) for the TEBP stoichiometry standards. Values with (triangles), and without (circles) calibration (cal) for RIKA efficiency are shown. In both cases, measured values matched well with the expected values with correlation coefficients of 0.997 (measured) and 0.998 (calibrated).

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