A Streamlined One-Pot Extraction Method Enables High-Sensitivity Top-Down Proteomic Characterization of Phospholamban from Biopsy-Scale Cardiac Tissue
TL;DR
A streamlined Azo-enabled one-pot extraction strategy for high-sensitivity top-down analysis of PLN that reduces the required tissue input to 1 mg while preserving analytical performance and establishing a practical strategy for TDP of PLN proteoforms from biopsy-scale and other sample-limited myocardial specimens.
Abstract
Top-down proteomics (TDP) enables comprehensive characterization of intact proteoforms, but the analysis of membrane proteoforms remains challenging because of their hydrophobicity and relatively low abundance. To address these challenges, we previously developed Azo, a photocleavable surfactant that enables efficient solubilization and top-down analysis of membrane proteins. Subsequently, we established an Azo-enabled TDP method permitting the comprehensive characterization of phospholamban (PLN), a transmembrane protein that plays crucial roles in calcium handling, directly from cardiac tissue. However, the original workflow required at least 10 mg of human cardiac tissue, making it unsuitable for biopsy-scale and other sample-limited studies. Herein, we developed a streamlined Azo-enabled one-pot extraction strategy for high-sensitivity top-down analysis of PLN that reduces the required tissue input to 1 mg while preserving analytical performance. Using online liquid chromatography–tandem mass spectrometry, we achieved highly reproducible detection, quantification, and post-translational modification localization of endogenous PLN proteoforms from 1 mg of human cardiac tissue. Notably, the streamlined strategy reduced the tissue required for PLN protein extraction 10-fold while retaining the same capacity for comprehensive characterization in the one-pot extraction. Collectively, these results establish a practical strategy for TDP of PLN proteoforms from biopsy-scale and other sample-limited myocardial specimens.
