Comprehensive Characterization of the Recombinant Catalytic Subunit of cAMP-Dependent Protein Kinase by Top-Down Mass Spectrometry
- 1 December 2019
- journal article
- research article
- Published by American Chemical Society (ACS) in Journal of the American Society for Mass Spectrometry
- Vol. 30 (12), 2561-2570
- https://doi.org/10.1007/s13361-019-02341-0
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.Keywords
Funding Information
- National Institutes of Health (R01 HL096971, R01 HL109810, R01 GM125085, R01 GM117058, S10 OD018475)
This publication has 55 references indexed in Scilit:
- Augmented Phosphorylation of Cardiac Troponin I in Hypertensive Heart FailureOnline Journal of Public Health Informatics, 2012
- An overview of enzymatic reagents for the removal of affinity tagsProtein Expression and Purification, 2011
- Mapping intact protein isoforms in discovery mode using top-down proteomicsNature, 2011
- Top-Down Quantitative Proteomics Identified Phosphorylation of Cardiac Troponin I as a Candidate Biomarker for Chronic Heart FailureJournal of Proteome Research, 2011
- Global Consequences of Activation Loop Phosphorylation on Protein Kinase AOnline Journal of Public Health Informatics, 2010
- Top-down high-resolution mass spectrometry of cardiac myosin binding protein C revealed that truncation alters protein phosphorylation stateProceedings of the National Academy of Sciences of the United States of America, 2009
- Decoding protein modifications using top-down mass spectrometryNature Methods, 2007
- Cell signalling in the cardiovascular system: an overviewPublished by BMJ ,2005
- Neurohormonal Activation in Congestive Heart Failure and the Role of VasopressinThe American Journal of Cardiology, 2005
- Targeting of Protein Kinase A by Muscle A Kinase-anchoring Protein (mAKAP) Regulates Phosphorylation and Function of the Skeletal Muscle Ryanodine ReceptorOnline Journal of Public Health Informatics, 2003