Abstract
The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.
Highlights
The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs)
Intra- and intermolecular interactions of sarcomeric thick and thin filament proteins are modifiable by PTMs, and it has been demonstrated that the intensity and dynamics of contraction and relaxation can be finely tuned via PTMs, those of thin filament proteins [2, 3]
Coomassie-stained bands shown in Fig. 3 represent 1⁄50–1⁄100 of the actual protein content harvested per OFFGEL electrophoresis (OGE) well
Summary
The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. RLC binds the S1-S2 lever arm of the myosin motor and is optimally positioned at the fulcrum to modulate interactions between the globular myosin heavy chain (MHC) head, the coiled coil light meromyosin thick filament backbone, and the neighboring MyBP-C (Fig. 1). Noncovalently bound ELC and RLC (i.e. the light chains of myosin) are more amenable to separation using standard biochemical methods due in part to their moderate sizes (22.4 and 18.9 kDa, respectively); because purification methods for either MHC or ELC/RLC are not time- and cost-effective, novel strategies for preparing/enriching these proteins in a single step are warranted
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