Top-Down Characterization of AKT3 Kinase by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

AKT protein kinases are vital serine/threonine kinases that function as central regulators of the PI3K/AKT signaling pathway, which controls various biological processes, including cell survival, growth, proliferation, and metabolism. Among the three AKT isoforms, AKT3 remains the least characterized, despite its involvement in brain malformations and cancers. Post-translational modifications (PTMs), particularly phosphorylation, regulate AKT function and activity, yet the proteoform landscape of AKT3 has not been characterized. Here, we performed top-down Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to characterize full-length recombinant human AKT3. Ultrahigh-resolution intact mass analysis resolved AKT3 proteoforms carrying up to three phosphorylations, with unphosphorylated and monophosphorylated species accounting for 40% and 39% of the total proteoform abundance, respectively. Complementary collisionally activated dissociation (CAD) and electron-capture dissociation (ECD) confirmed the removal of the initiating methionine and localized the N-terminal acetylation. Targeted tandem mass spectrometry (MS/MS) of monophosphorylated AKT3 generated terminal and internal fragments that localized the primary phosphorylation site to Thr332 (canonical Thr305). These findings uncover AKT3 proteoform diversity and demonstrate the power of ultrahigh-resolution top-down FTICR-MS for resolving and characterizing intact kinase proteoforms.