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

ABSTRACT: 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.

INSTRUMENT(S): impact II, maXis, solariX, nanoACQUITY UPLC

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Ying Ge   Holden Rogers  

PROVIDER: MSV000091522 | MassIVE | Tue Mar 21 14:17:00 GMT 2023

SECONDARY ACCESSION(S): PXD041013

REPOSITORIES: MassIVE