Ultrasensitive and absolute quantification of the phosphoinositide 3-kinase/Akt signal transduction pathway by mass spectrometry

Abstract

The phosphoinositide 3-kinase (PI3K)/Akt pathway controls a vast array of normal physiological processes and is frequently aberrantly activated in cancer, thus identifying PI3K/Akt-signaling components as promising drug targets in oncology. However, implementation of rational cancer therapies for this pathway needs robust and simple tools to stratify patients according to PI3K pathway activation and to validate and measure the impact of targeted inhibition on primary cancer tissues. Herein we present a technique for the quantification of the PI3K/Akt-signaling pathway based on the mass spectrometric measurement of PI3K-dependent protein kinase activity in cell lysates. The concept of this application of MS is to exploit enzymatic activity to amplify the signal of the enzyme under study analogous to the PCR used to amplify nucleic acid sequences. We show that this approach allows quantitative analysis of a cell-signaling pathway with high sensitivity, precision of quantification, and specificity. Due to its special analytical capabilities and potential for multiplexing, this approach could contribute significantly to cell-signaling studies and to the development and implementation of personalized cancer therapies.

Fig. 1.

Principle and precision of mass spectrometric quantification of protein kinase activity. (A) Principle of the method. (B) Precision of the method. Known amounts of synthetic p-Aktide were mixed with a fixed amount of IS peptide and analyzed by MALDI-TOF MS, LC-MS, or LC-MS/MS. Functions derived from standard curves (see Upper for an example) were used to recalculate the concentration of added p-Aktide and thus estimate coefficients of variation (CV) (Lower).

Fig. 2.

Sensitivity of Aktide kinase activity quantification by MS. (A) Quantification of Aktide kinase activity by using recombinant Akt/PKB. Decreasing amounts of recombinant Akt/PKB were used in activity assays with Aktide as a substrate. (Lower) Shown is a MALDI-TOF MS spectrum obtained by using 0.02 pg of Akt, which corresponds to ≈500 zmol (500 × 10⁻²¹ mol) of protein. S/N, signal-to-noise ratio. (B) Quantification of Aktide kinase activity in cell lysates. (Upper) Representative chromatograms from LC-MS runs obtained by using different amounts of WEHI-231 B cell lysate (gray and black lines correspond to IS and p-Aktide signals, respectively). (Lower) Shown is a plot of the quantitative data derived from these chromatograms.

Fig. 3.

Specificity of Aktide kinase activity for the PI3K/Akt pathway. (A) Specificity in cell lysates. Cells were treated with different concentrations of wortmannin (WM, a pan PI3K inhibitor) or IC87114 (a PI3K inhibitor with selectivity for the p110δ isoform of PI3K) before lysis and MS activity measurements. (B) Aktide kinase activity in total cell lysates and Akt/PKB immunoprecipitates (IP) from untreated cells were equally sensitive to WM pretreatment. (C) Shown is kinase activity in WEHI-231 lysates at different in vitro kinase reaction times. Activities increased upon stimulation with PI3K/Akt pathway agonists [anti-IgM (Bottom) or pervanadate (Middle)] in a WM-sensitive manner. (Top) Shown is a Western blot of total cell lysates with anti-p-Akt/PKB (p-Ser-473) antibodies, demonstrating a correlation between Aktide kinase activity and p-Akt/PKB measurements.

Fig. 4.

Quantification of PI3K/Akt pathway activity in solid and liquid tumors. (A) B16/Bl6 solid tumors in mice were treated with DMSO (vehicle) or LY294002 (a pan-PI3K inhibitor) before excision and MS activity measurements. (B) Primary acute myeloid leukemia cells were sorted into CD34⁺CD38⁻ stem cell and CD34⁺CD38⁺ bulk tumor fractions, and activity toward Aktide was measured by MS.