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Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry

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.

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Acknowledgements

The authors would like to thank Dr. Wenxuan Cai for preparing the construct for PKA C-subunit for bacterial expression. Financial support was provided by NIH R01 GM117058 (to S. J. and Y. G.) and R01 GM125085 (to Y. G.). Y. G. also would like to acknowledge the NIH grants, R01 HL096971, R01 HL109810, and S10 OD018475.

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Correspondence to Ying Ge.

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Wu, Z., Jin, Y., Chen, B. et al. Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry. J. Am. Soc. Mass Spectrom. 30, 2561–2570 (2019). https://doi.org/10.1007/s13361-019-02341-0

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Keywords

  • Post-translational modifications
  • Top-down mass spectrometry
  • Protein kinases