Solubilization, two-dimensional separation and detection of the cardiac myofilament protein troponin T.
Proteomics strongly relies on the separation of complex protein mixtures, with two-dimensional electrophoresis (2-DE) being a commonly used technique. However efficient separation requires adequate solubilization of the original sample which will determine whether all proteins are accurately represented. Cardiac muscle has presented a particular challenge to solubilization. Here we have optimized the solubilization, separation and detection of the myofilament protein troponin T (TnT). Human left ventricular tissue from a rejected donor transplant heart was homogenized under 19 different conditions and subjected to 2-DE and Western blot analysis for TnT. The optimal conditions for isoelectric focusing of intact TnT requires homogenization in 6 M urea, 2.5 M thiourea, 4% 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate, with the addition of NaCl (2.5 M final concentration). TnT degradation products present in this severely damaged heart however, were differentially extracted from both each other and the intact molecule under the various conditions used. Despite adequate focusing of TnT it was found that a nonglutaraldehyde silver staining protocol, that is compatible with subsequent mass spectrometry, has greatly reduced sensitivity for TnT compared to Coomassie blue. Thus, care is required to avoid misrepresentation of troponin T in proteomic analysis in cardiac tissue.
Department of Physiology, Queen' University Kingston, Ontario, Canada.
Ralf
R
Labugger
Jason L
JL
McDonough
Irina
I
Neverova
Jennifer E
JE
Van Eyk
D015153:Blotting, Western; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D016027:Heart Transplantation; D006352:Heart Ventricles / Q000473:pathology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D009206:Myocardium / Q000166:cytology*; D020107:Troponin T / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D020107:Troponin T