Liquid-phase isoelectric focusing of cardiac proteins: differential separation of cMyBP-C phosphorylation states

Abstract

Cardiac myosin binding protein C (cMyBP-C) has three phosphorylation sites in its N-terminus (Ser273, Ser282, and Ser302), which are assumed to contribute to the regulation of thick filament spacing and function. We adopted a 2-D technique consisting of a liquid-phase isoelectric focusing (IEF) in a 5–7 pI gradient using the Rotofor system (Biorad). Ten fractions of 0.2-pH intervals were obtained, separated by SDS-PAGE and further analyzed by immunoblotting for both total cMyBP-C and the Ser282-phospho-cMyBP-C (P-cMyBP-C). Mouse hearts were challenged with 1 μM isoprenaline for 10 min in a Langendorff perfusion system. Coomassie staining of the electrophoretically separated 10 IEF-fractions revealed a differential protein expression pattern in each fraction, with the expected highest amount at the neutral pH. Total cMyBP-C of control hearts was detected in 3 fractions at the full size of 150 kDa. The theoretical pI of the fractions was 6.4, 6.6 and 6.8. The majority of cMyBP-C was in the first two fractions. P-cMyBP-C was highest in the 6.4 pI fraction. Isoprenaline treatment induced a shift of cMyBP-C to the 6.2 pI fraction, which was also P-cMyBP-C positive. Concomitantly cMyBP-C was reduced in the 6.6 and 6.8 pI fractions. IEF of neonatal rat cardiomyocytes (NRCM) using a 3–10 pI gradient identified cMyBP-C in the 6.5 pI fraction. In conclusion, this report shows that the phosphorylation states of cMyBP-C from adult mouse ventricle or NRCM can be separated using liquid-phase IEF. Furthermore, the IEF fractions yielded by this approach allow multiple analyses of a single fraction including mass-spectrometry.