Subfractionation of heart tissue: the "in sequence" myofilament protein extraction of myocardial tissue
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Quantitative Analysis of Chromatin Proteomes in Disease
Published on: December 28, 2012
Lesley A Kane1, Irina Neverova, Jennifer E Van Eyk
1Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA.
Insights
This study presents a heart tissue fractionation protocol to separate abundant myofilament proteins. This method enables in-depth proteomic analysis of lower-abundance proteins using standard techniques.
Area of Science:
- Biochemistry
- Proteomics
- Cardiovascular Research
Background:
- Proteomic analysis of heart tissue is challenging due to the wide dynamic range of protein concentrations.
- Highly abundant myofilament proteins can mask the detection of low-abundance proteins.
Purpose of the Study:
- To develop a robust protocol for fractionating heart tissue.
- To facilitate the proteomic analysis of low-abundance proteins in cardiac samples.
Main Methods:
- A novel protocol for heart tissue fractionation based on protein solubility at different pH levels.
- Extraction of cytoplasmic, myofilament, and membrane protein fractions.
- Compatibility with established proteomics technologies like 2D electrophoresis and HPLC.
Main Results:
- Successful separation of heart tissue proteins into three distinct fractions: cytoplasmic-enriched, myofilament-enriched, and membrane protein-enriched.
- The protocol effectively isolates abundant myofilament proteins, reducing their interference in subsequent analyses.
- The fractionation method is compatible with standard proteomic workflows.
Conclusions:
- This fractionation strategy significantly enhances the depth and accuracy of cardiac proteomic studies.
- The protocol provides a foundation for comprehensive analysis of cardiac proteomes.
- It enables the identification and quantification of low-abundance cardiac proteins previously obscured by abundant ones.
Abstract:
Proteomic analysis of heart tissue is complicated by the large dynamic range of its proteins. The most abundant proteins are the myofilament proteins, which comprise the contractile apparatus. This chapter describes a protocol for fractionation of heart tissue that extracts the myofilament proteins into a separate sample fraction, allowing analysis of lower-abundance proteins. Importantly, this is performed in a manner that is compatible with two-dimensional electrophoresis and high-performance liquid chromatography, two of main technologies of proteomics. The method produces three fractions based on solubility at different pHs: (1) cytoplasmic-enriched extract (neutral pH), (2) myofilament-enriched extract (acidic pH), and (3) membrane protein-enriched pellet. Fractionation of heart tissue in this manner provides the basis for in-depth proteomic analysis.
