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 reversephase 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.

Obesity is associated with diminished antibody production against influenza infection. Therefore, therapeutic strategies are needed to enhance humoral immunity. We previously demonstrated that essential long-chain n-3 polyunsaturated fatty acids (PUFA) enhance humoral responses to a T-independent antigen. Thus, here we hypothesized that the n-3 PUFA docosahexaenoic acid (DHA) could improve antibody production upon influenza infection in obese mice. To test the hypothesis, mice were fed for 15 weeks with a control or a Western diet with or without DHA supplementation followed by infection with influenza A/Puerto Rico/8/34. The data show that DHA supplementation improved antibody titers, as measured by HAI and microneutralization assays, in obese mice after influenza infection. Mechanistically, DHA did not target B-cell PPARγ or the DHA-sensing G-protein coupled receptor 120 to boost antibody levels. Instead, DHA increased the levels of downstream D-series specialized pro-resolving lipid mediators (SPMs), which are known to increase antibody production through the generation of CD138+ antibody secreting cells. Specifically, LC/MS analyses showed 14-HDHA to be elevated upon DHA intervention, which elevated antibody levels upon infection and increased the frequency of CD138+ cells. Overall, the results suggest that DHA may have potential therapeutic applications for improving humoral immunity, potentially through an SPM mediated mechanism.