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). In particular, membrane proteins play critical roles in cellular functions and represent the largest class of drug targets. However, the top-down MS characterization of endogenous membrane proteins remains challenging, mainly due to their intrinsic hydrophobicity and low abundance. Phospholamban (PLN) is a regulatory membrane protein located in the sarcoplasmic reticulum and is essential for regulating cardiac muscle contraction. PLN has diverse combinatorial PTMs and their dynamic regulation has 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 employed a two-pronged top-down MS approach using an online reversed-phase liquid chromatography tandem MS (LC-MS/MS) method on a quadrupole time-of-flight (Q-TOF) MS and a direct infusion method via an ultrahigh-resolution Fourier-transform ion cyclotron resonance (FTICR) MS. We have comprehensively characterized the sequence and combinatorial PTMs of endogenous human cardiac PLN. We have shown the site-specific localization of phosphorylation to Ser16 and Thr17 by MS/MS for the first time and the localization of S-palmitoylation to Cys36. Taken together, we have developed a streamlined top-down targeted proteomics method for comprehensive characterization of combinatorial PTMs in PLN toward better understanding the role of PLN in cardiac contractility.