A Streamlined One-Pot Extraction Method Enables High-Sensitivity Top-Down Proteomic Characterization of Phospholamban from Biopsy-Scale Cardiac Tissue.
6 affiliations
- 1Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin53706, United States.
- 2Department of Cell and Regenerative Biology, University of Wisconsin-Madison, Madison, Wisconsin53705, United States.
- 3Department of Animal and Dairy Sciences, University of Wisconsin-Madison, Madison, Wisconsin53706, United States.
- 4Department of Medicine-Cardiovascular Disease, University of Wisconsin-Madison, Madison, Wisconsin53705, United States.
- 5Cardiovascular Research Center, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin53706, United States.
- 6Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin53706, United States.
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.
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BibTeX
@article{clemmer2026streamlined,
title = {{A Streamlined One-Pot Extraction Method Enables High-Sensitivity Top-Down Proteomic Characterization of Phospholamban from Biopsy-Scale Cardiac Tissue.}},
author = {Isabella R Clemmer and Holden T Rogers and Mallory C Wilson and Zhan Gao and Hsin-Ju Chan and Zachery R Gregorich and Farhan Raza and Ying Ge},
journal = {Journal of the American Society for Mass Spectrometry},
year = {2026},
month = oct,
doi = {10.1021/jasms.6c00309},
url = {https://europepmc.org/article/MED/42916765},
note = {CC BY},
}
RIS
TY - JOUR
TI - A Streamlined One-Pot Extraction Method Enables High-Sensitivity Top-Down Proteomic Characterization of Phospholamban from Biopsy-Scale Cardiac Tissue.
AU - Isabella R Clemmer
AU - Holden T Rogers
AU - Mallory C Wilson
AU - Zhan Gao
AU - Hsin-Ju Chan
AU - Zachery R Gregorich
AU - Farhan Raza
AU - Ying Ge
PY - 2026
DA - 2026/10/01
JO - Journal of the American Society for Mass Spectrometry
DO - 10.1021/jasms.6c00309
UR - https://europepmc.org/article/MED/42916765
AB - 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.
SN - 1044-0305
SN - 1879-1123
ER -