Figure 2.
Optimization of a RapidFire mass spectrometry (MS) assay. (A), RapidFire MS assay scheme for quantification of the dephosphorylated peptide product from the WIP1 reaction. (B), The integrated areas from MS analysis of the heavy-labeled product peptide 4 are linearly related to peptide concentrations between 0 and 2.5 μm (R2 = 0.94), which serves as a calibration standard for the WIP1 reaction product. (C), A range of WIP1 reaction product peptide concentrations were analyzed and the limit of quantitation was determined to be 28.3 nm. (D), The optimized assay is robust with an 80-fold signal-to-background value between the vehicle (DMSO) and 100% inhibition (GSK2830371) controls and a Z′-factor value of 0.74. Individual data points are shown and bars indicate mean ± S.D. (n = 144). (E), Titrations of DMSO demonstrated that assay performance is not diminished by increased vehicle concentrations up to 1.9% (mean ± S.D.; n = 16). (F), Initial velocities for WIP1 with a range of P53 phosphopeptide substrate 3 concentrations as determined by Rapidfire MS. (G), Velocities measured from the data in (F) were plotted and fit to the Michaelis-Menten equation to estimate the Km at 1.8 μm.