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A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample
Published on: June 1, 2017
Nonionic detergent phase extraction for the proteomic analysis of heart membrane proteins using label-free LC-MS
Pamela M Donoghue1, Chris Hughes, Johannes P C Vissers
1Proteome Research Centre, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland. pamela.donoghue@gmail.com
Insights
This study validates a new method to isolate cardiac membrane proteins, aiding the discovery of heart failure biomarkers. The technique enriches specific protein subsets for more effective analysis in heart disease research.
Area of Science:
- Biochemistry
- Cardiovascular Research
- Proteomics
Background:
- Heart failure, a major cause of mortality, stems from systemic or specific heart muscle diseases like dilated cardiomyopathy (DCM).
- Proteome-wide studies often miss crucial biomarkers due to sample complexity.
- Subproteome analysis offers a focused approach to identify disease-specific markers.
Purpose of the Study:
- To validate a Triton X-114-based phase enrichment method for isolating cardiac membrane proteins.
- To assess the efficacy of nanoscale liquid chromatography-mass spectrometry (LC-MS) for analyzing cardiac subproteomes.
- To demonstrate a novel approach for discovering heart failure biomarkers.
Main Methods:
- Applied label-free nanoscale LC-MS to analyze protein samples.
- Utilized a Triton X-114-based phase enrichment technique to isolate membrane proteins.
- Performed annotation of subcellular location and GRAVY score analysis for protein separation.
Main Results:
- Achieved over 62% enrichment of cardiac membrane proteins.
- Successfully separated soluble and membrane-bound proteins.
- Confidently identified and annotated hydrophobic proteins in a control sample.
Conclusions:
- The validated Triton X-114 method effectively enriches cardiac membrane proteins.
- Nanoscale LC-MS combined with this enrichment technique is powerful for identifying integral membrane proteins.
- This approach holds promise for future studies on cardiac membrane subproteome changes in heart disease.
Abstract:
Heart diseases resulting in heart failure are among the leading causes of morbidity and mortality in the Western world and can result from either systemic disease (e.g., hypertensive heart disease, ischemic heart disease) or specific heart muscle disease (e.g., dilated cardiomyopathy/DCM). Subproteome analysis of such disease subsets affords a reduction in sample complexity, potentially revealing biomarkers of cardiac failure that would otherwise remain undiscovered in proteome wide studies. Label-free nanoscale LC-MS has been applied in this study to validate a Triton X-114-based phase enrichment method for cardiac membrane proteins. Annotation of the subcellular location combined with GRAVY score analysis indicates a clear separation between soluble and membrane-bound proteins with an enrichment of over 62% for this protein subset. LC-MS allowed confident identification and annotation of hydrophobic proteins in this control sample pilot study and demonstrates the power of the proposed technique to extract integral membrane-bound proteins. This approach should be applicable to a wider scale study of disease-associated changes in the cardiac membrane subproteome.
