Abstract
Reversible phosphorylation plays critical roles in cell growth, division, and signal transduction. Kinases which catalyze the transfer of γ-phosphate groups of nucleotide triphosphates to their substrates are central to the regulation of protein phosphorylation and are therefore important therapeutic targets. Top-down mass spectrometry (MS) presents unique opportunities to study protein kinases owing to its capabilities in comprehensive characterization of proteoforms that arise from alternative splicing, sequence variations, and post-translational modifications. Here, for the first time, we developed a top-down MS method to characterize the catalytic subunit (C-subunit) of an important kinase, cAMP-dependent protein kinase (PKA). The recombinant PKA C-subunit was expressed in Escherichia coli and successfully purified via his-tag affinity purification. By intact mass analysis with high resolution and high accuracy, four different proteoforms of the affinity-purified PKA C-subunit were detected, and the most abundant proteoform was found containing seven phosphorylations with the removal of N-terminal methionine. Subsequently, the seven phosphorylation sites of the most abundant PKA C-subunit proteoform were characterized simultaneously using tandem MS methods. Four sites were unambiguously identified as Ser10, Ser11, Ser18, and Ser30, and the remaining phosphorylation sites were localized to Ser2/Ser3, Ser358/Thr368, and Thr[215-224]Tyr in the PKA C-subunit sequence with a 20mer 6xHis-tag added at the N-terminus. Interestingly, four of these seven phosphorylation sites were located at the 6xHis-tag. Furthermore, we have performed dephosphorylation reaction by Lambda protein phosphatase and showed that all phosphorylations of the recombinant PKA C-subunit phosphoproteoforms were removed by this phosphatase.