Application of reversed phase high performance liquid chromatography for subproteomic analysis of cardiac muscle.

The application of protein separation methodologies, such as reversed phase chromatography, should allow differential separation of the proteome, or at least specific subproteomes, comparable to that achieved by two-dimensional electrophoresis (2-DE). A rapid sequential protein extraction method (termed "IN Sequence") was developed to isolate three distinct subproteomes of cardiac muscle. Two subproteomes, those enriched for the cytoplasmic or myofilament proteins, can be separated by either reversed phase high performance liquid chromatography (RP-HPLC) or 2-DE. Reversed phase HPLC of the myofilament protein enriched extract was optimized for resolution and peak numbers by altering flow rate, gradient rate and the organic modifiers, isopropanol and acetronitrile. The myofilament protein enriched extract from failing swine heart, due to coronary artery ligation (LAD), was compared to the extract from a sham operated animal (SHAM). The HPLC chromatograms of these extracts were similar, but distinctive in many regions. The HPLC fractions, collected within some of these distinct regions of the chromatograms were analyzed using peptide mass fingerprinting - mass spectrometry and immunoblot analysis. Two myofilament proteins, troponin T and myosin heavy chain, were identified and found differentially modified in the SHAM and LAD hearts. Both troponin T and myosin heavy chain are problematic proteins for 2-DE, but yet they were resolved by reversed phase chromatography. Therefore, RP-HPLC can be used in conjunction with 2-DE to enhance protein separation of myofilament protein subproteome.