TY - JOUR
T1 - Solubilization, two-dimensional separation and detection of the cardiac myofilament protein troponin T
AU - Labugger, Ralf
AU - McDonough, Jason L.
AU - Neverova, Irina
AU - Van Eyk, Jennifer E.
PY - 2002/7/8
Y1 - 2002/7/8
N2 - 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.
AB - 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.
KW - Cardiac muscle
KW - Solubilization
KW - Troponin T
KW - Two-dimensional gel electrophoresis
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U2 - 10.1002/1615-9861(200206)2:6<673::AID-PROT673>3.0.CO;2-2
DO - 10.1002/1615-9861(200206)2:6<673::AID-PROT673>3.0.CO;2-2
M3 - Article
C2 - 12112847
AN - SCOPUS:0035987493
SN - 1615-9853
VL - 2
SP - 673
EP - 678
JO - Proteomics
JF - Proteomics
IS - 6
ER -