Dilated cardiomyopathy affects 4.5 cases per 100,000 population annually, based on autopsy findings. Clinically, 2.45 cases per 100,000 population are detected annually.1
In dilated cardiomyopathy the left ventricle enlarges, followed by the right ventricle and then the atria. Untreated or inadequately treated, it can eventually lead to heart failure. Symptoms include shortness of breath, fatigue, and swelling of the ankles, feet, legs, abdomen and veins in the neck.
Somewhat recently, mass spectrometry-based proteomic studies have been able to comprehensively identify disease-associated alterations in the cardiac proteome. The proteolytically digested protein samples are fractionated using high-performance liquid chromatography (HPLC) and subjected to ionization and mass analysis.2,3 However, researchers were unable to fully quantify proteins from minute quantities of human tissues using peptide or protein-labelling approaches such as isobaric tags for relative and absolute quantitation (iTRAQ) or isotope-coded protein labelling (ICPL).4,5 Researchers have, therefore, refined the methodology to perform the quantitative proteome analysis of heart biopsies using limited quantities of tissue.6
Elke Hammer and colleagues from the Interfakultäres Institut für Genetik und Funktionelle Genomforschung, Universitätsmedizin Greifswald in Germany used an initial 1–2 mg of tissue material for the proteomic analysis. They digested the crude protein extracts with LysC and trypsin, followed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) using a high-resolution linear ion trap coupled to a Fourier transform ion cyclotron resonance (LTQ FTICR) mass spectrometer (Thermo Electron Corp., Bremen, Germany). To reduce sampling errors, the researchers used protein extracts from multiple biopsies per patient.
The researchers used Rosetta Elucidator software to quantify the detected MS signals from all the individual LC-MS/MS analyses. The researchers explain that such an approach enables quantitation of low-abundant proteins in muscle tissue, by assigning MS/MS events in runs with higher-intensity signals to low-intensity signals of other runs. The protein analysis through evolutionary relationships classification software (PANTHER, www.pantherdb.org) facilitates the prediction of biological processes or molecular function of differentially expressed proteins. Protein annotation with the PANTHER software revealed substantial differences in abundance of mitochondrial (34.5%) and cytoskeletal (24.7%) proteins. Metabolic pathways were affected as well.
“The large number of proteins involved in energy-providing pathways which displayed decreased intensity in dilated cardiomyopathy points to a limited energy supply in the myocardium,” the researchers conclude. The elevated levels of structural proteins point to impaired cellular integrity.
- Rakar, S., et al. (1997) “Epidemiology of dilated cardiomyopathy: A prospective post-mortem study of 5252 necropsies. The Heart Muscle Disease Study Group,” European Heart Journal, 18(1) (pp. 117-23).
- Kislinger, T., et al. (2005) “Multidimensional protein identification technology (MudPIT): Technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue,” Journal of the American Society for Mass Spectrometry, 16 (pp. 1207–1220).
- Kline, K.G., and Wu, C.C. (2009) “MudPIT analysis: Application to human heart tissue,” Methods in Molecular Biology (Clifton, NJ), 528 (pp. 281–293), doi: 10.1007/978-1-60327-310-7_20.
- Ross, P.L., et al. (2004) “Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents,” Molecular & Cellular Proteomics, 3, (pp. 1154–1169), doi: 10.1074/mcp.M400129-MCP200.
- Flory, M.R., et al. (2002) “Advances in quantitative proteomics using stable isotope tags,” Trends in Biotechnology, 20 (pp. S23–S29).
- Hammer, E., et al. (2013) “Characterization of the human myocardial proteome in dilated cardiomyopathy by label-free quantitative shotgun proteomics of heart biopsies,” Methods in Molecular Biology, 1005 (pp. 67-76), doi: 10.1007/978-1-62703-386-2_6.
Post Author: Sridhar Nadamuni.