Solubilization, two-dimensional separation and detection of the cardiac myofilament protein troponin T

Abstract

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.

Multidimensional protein identification technology (MudPIT): Technical overview of a profiling method optimized for the comprehensive proteomic investigation of normal and diseased heart tissue

“…Western blot analysis of mitochondrial markers, such as the ␤-subunit of the membrane-associated F 1 -ATPase, generates very strong signals°in°the°nuclear°(and°all°other)°fractions°(Figure°3d; top panel). This cross-contamination was not entirely unexpected, given the highly specialized biomechanical adaptations of muscle, and has been reported previously by van Eyk and colleagues in 2D-gel proteomic studies°of°human°cardiac°tissue° [36].°Nevertheless,°it proved to be particularly debilitating for detection of proteins of biological interest. In fact, contaminating contractile apparatus (e.g., ventricular myosins) and even blood proteins (hemoglobins) were detected in nuclear extracts prepared from cardiac myocyte nuclei using a standard two-step centrifugation protocol following vigorous homogenization of heart tissue with an electrical blade (Polytron) [data not shown].…”

Strategy for Analysis of Cardiac Troponins in Biological Samples with a Combination of Affinity Chromatography and Mass Spectrometry

“…The extreme pI values for cTnC (4.05) and cTnI (9.87), the strong binding affinity between the subunits, and the poor silver staining properties present a particular challenge to cardiac troponin analysis by a traditional proteomic approach, i.e., protein separation with two-dimensional gel electrophoresis, silver staining, and in-gel digestion for PMF by mass spectrometry (31 ). In comparison, the advantages of the affinity-chromatography/onbead digestion approach, performed in a single microtube, are numerous.…”

Chronic xanthine oxidase inhibition prevents myofibrillar protein oxidation and preserves cardiac function in a transgenic mouse model of cardiomyopathy

“…After the pellet was resuspended and homogenized twice using the buffer above, the pellet was extracted using an acid extraction buffer containing 0.02% trifluoroacetic acid and 1 mM tris(2-carboxyethyl)phosphine hydrochloride. Extraction with this buffer results in a myofilament-enriched fraction (18).…”