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

Top-down mass spectrometry (MS)-based proteomics has
become a powerful
tool for analyzing intact proteins and their associated post-translational
modifications (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 method
on a quadrupole time-of-flight MS and a direct infusion method via
an ultrahigh-resolution Fourier-transform ion cyclotron resonance
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. Moreover,
we applied our method to characterize PLN in disease and reported
the significant reduction of PLN phosphorylation in human failing
hearts with ischemic cardiomyopathy. 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.