A Novel, In-Solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain
journal contributionposted on 30.04.2012, 00:00 authored by Sarah B. Scruggs, Rick Reisdorph, Mike L. Armstrong, Chad M. Warren, Nichole Reisdorph, R. John Solaro, Buttrick Buttrick
The molecular conformation of the cardiac myosin motor is modulated by inter-molecular interactions among the heavy chain, the light chains, myosin binding protein-C (MyBP-C) and titin, and is governed by post-translational modifications (PTMs). In-gel digestion followed by liquid chromatography mass spectrometry (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 workflow 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. Sub-cellular fractionation followed by OFFGEL electrophoresis (OGE) resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain (MHC), and MyBPC of the thick filament. Further purification of RLC using reverse phase (RP) -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 OGE fractions were un-phosphorylated (78.8%), singly- (18.1%) and doubly-phosphorylated (3.1%) RLC. The novel aspects of this study are: 1) milligram amounts of endogenous cardiac sarcomeric sub-proteome were focused with resolution comparable to 2DE, 2) separation and quantification of post-translationally modified variants was achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in-solution, 4) endogenous charge variants of RLC were separated; a novel doublyphosphorylated 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.