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

Top-down mass spectrometry (MS)-based proteomics hasbecome a powerfultool for analyzing intact proteins and their associated post-translationalmodifications (PTMs). In particular, membrane proteins play criticalroles in cellular functions and represent the largest class of drugtargets. However, the top-down MS characterization of endogenous membraneproteins remains challenging, mainly due to their intrinsic hydrophobicityand low abundance. Phospholamban (PLN) is a regulatory membrane proteinlocated in the sarcoplasmic reticulum and is essential for regulatingcardiac muscle contraction. PLN has diverse combinatorial PTMs, andtheir dynamic regulation has significant influence on cardiac contractilityand disease. Herein, we have developed a rapid and robust top-downproteomics method enabled by a photocleavable anionic surfactant,Azo, for the extraction and comprehensive characterization of endogenousPLN from cardiac tissue. We employed a two-pronged top-down MS approachusing an online reversed-phase liquid chromatography tandem MS methodon a quadrupole time-of-flight MS and a direct infusion method viaan ultrahigh-resolution Fourier-transform ion cyclotron resonanceMS. We have comprehensively characterized the sequence and combinatorialPTMs of endogenous human cardiac PLN. We have shown the site-specificlocalization of phosphorylation to Ser16 and Thr17 by MS/MS for thefirst time and the localization of S-palmitoylation to Cys36. Moreover,we applied our method to characterize PLN in disease and reportedthe significant reduction of PLN phosphorylation in human failinghearts with ischemic cardiomyopathy. Taken together, we have developeda streamlined top-down targeted proteomics method for comprehensivecharacterization of combinatorial PTMs in PLN toward better understandingthe role of PLN in cardiac contractility.

Automated summary

We have developed a rapid and robust top-down proteomics method to extract and comprehensively characterize endogenous PLN from cardiac tissue, enabling a better understanding of its role in cardiac contractility.