MassIVE MSV000091522

Partial Public PXD041013

Comprehensive Characterization of Endogenous Phospholamban Proteoforms Enabled by Photocleavable Surfactant and Top-down Proteomics

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Description

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for analyzing intact proteins and their associated post-translational modification (PTMs). Membrane proteins play critical roles in numerous cellular functions and represent the largest class of drug targets. However, top-down MS characterization of endogenous membrane proteins remains challenging, mainly due to their hydrophobicity and low expression level. Phospholamban (PLN) is a regulatory membrane protein in the sarcoplasmic reticulum and is essential in controlling cardiac contraction through calcium handling. Endogenously, the dynamic regulation of PLN's combinatorial PTM state has a significant influence on cardiac contractility and disease. Herein, we have developed a rapid and robust top-down proteomics method enabled by a photocleavable anionic surfactant, Azo, for the extraction and comprehensive characterization of endogenous PLN from cardiac tissue. We have employed a two-pronged top-down MS approach utilizing an online reversed-phase liquid chromatography tandem MS (LC-MS/MS) on a quadrupole time-of-flight MS and an ultrahigh-resolution Fourier-transform ion cyclotron resonance (FTICR) MS via direct infusion. We have comprehensively characterized the sequence and combinatorial PTMs of endogenous PLN, including S-palmitoylation and phosphorylations. S-palmitoylation was localized to Cys36 and for the first time with MS/MS, we have successfully localized the site-specific phosphorylation to Ser16 and Thr17 in human PLN extracted directly from heart tissue. We envision this top-down proteomics method will be crucial in its application to understanding PLN's role in cardiac contractility. [doi:10.25345/C5RB6WC0R] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Phospholamban ; Top-down Proteomics ; Azo

Contact

Principal Investigators:
(in alphabetical order) 		Holden Rogers, University of Wisconsin-Madison, United States of America
Ying Ge, University of Wisconsin-Madison, United States of America
Submitting User: htrogers
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