Distinct sequences and post-translational modifications in cardiac atrial and ventricular myosin light chains revealed by top-down mass spectrometry

Abstract

Myosin is the principal component of the thick filaments that, through interactions with the actin thin filaments, mediates force production during muscle contraction. Myosin is a hexamer, consisting of two heavy chains, each associated with an essential (ELC) and a regulatory (RLC) light chain, which bind the lever-arm of the heavy chain and play important modulatory roles in striated muscle contraction. Nevertheless, a comprehensive assessment of the sequences of the ELC and RLC isoforms, as well as their post-translational modifications, in the heart remains lacking. Herein, utilizing top-down high-resolution mass spectrometry (MS), we have comprehensively characterized the sequences and N-terminal modifications of the atrial and ventricular isoforms of the myosin light chains from human and swine hearts, as well as the sites of phosphorylation in the swine proteins. In addition to the correction of disparities in the database sequences of the swine proteins, we show for the first time that, whereas the ventricular isoforms of the ELC and RLC are methylated at their N-termini, which is consistent with previous studies, the atrial isoforms of the ELC and RLC from both human and swine are Nα-methylated and Nα-acetylated, respectively. Furthermore, top-down MS with electron capture dissociation enabled localization of the sites of phosphorylation in swine RLC isoforms from the ventricles and atria to Ser14 and Ser22, respectively. Collectively, these results provide new insights into the sequences and modifications of myosin light chain isoforms in the human and swine hearts, which will pave the way for a better understanding of their functional roles in cardiac physiology and pathophysiology.

Study on the transcriptome for breast muscle of chickens and the function of key gene RAC2 on fibroblasts proliferation

“…Myosin light chain ( MyLC ) can be further divided into essential myosin light chain ( ELC ) and regulatory myosin light chain ( RLC ), which are encoded by a variety of genes, including MYL1, MYL2, MYL3 , etc. [ 34 ]. It is generally believed that the main function of ELC is to maintain the configuration of heavy chain, while RLC plays a role in regulating the activity of muscle fiber, and the proportion of different types for myosin light chains would affect the type and growth of muscle fiber [ 35 ].…”

Reproducible large-scale synthesis of surface silanized nanoparticles as an enabling nanoproteomics platform: Enrichment of the human heart phosphoproteome

“…S8b,d). Furthermore, using this integrated top-down phosphoproteomics strategy, we have enriched, detected and examined multiple known cardiac phosphoproteins that were previously undetected or in extremely low abundance in the HEPES cardiac tissue extraction [31,60,61]: phosphorylated chromobox protein homolog 1 (CBX1; M r 21755.7; Fig. 5c), phosphorylated tropomyosin alpha (TPM1; M r 32809.7; Fig.…”

Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics

“…Moreover, although the conventional bottom- up approach is capable of identifying and quantifying thousands of proteins from a complex mixture [71, 72], due to limited sequence coverage as a result of proteolytic digestion, it remains challenging to distinguish highly homologous protein isoforms and proteolytic products with small truncation. On the contrary, top-down MS-based proteomics approach permits the analysis of intact proteins and unambiguously reveals the existing proteoforms including those with small truncation <1000 Da, as well as those with distinct sequence variations [73]. In addition, high-resolution top-down MS allows for the determination of accurate molecular weight with less than 10 ppm discrepancy.…”