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. 2017 Jun:107:13-21.
doi: 10.1016/j.yjmcc.2017.04.002. Epub 2017 Apr 17.

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

Zachery R Gregorich  1 , Wenxuan Cai  1 , Ziqing Lin  2 , Albert J Chen  2 , Ying Peng  2 , Takushi Kohmoto  3 , Ying Ge  4
Affiliations

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

Zachery R Gregorich et al. J Mol Cell Cardiol. 2017 Jun.

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.

Keywords: Acetylation; Methylation; Myosin light chain; Phosphorylation; Post-translational modification; Top-down mass spectrometry.

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Figures

Fig. 1
Fig. 1
Overview of top-down MS strategy for the identification and characterization of myosin light chain isoforms in the atria and ventricles of the heart. (I) Extraction of the myofilament sub-proteome from atrial and ventricular tissue. (II) 1DLC separation of myofilament sub-proteomes and fraction collection of myosin light chain isoforms. (III) MS profiling of myosin light chain isoforms and the quantification of RLC phosphorylation. (IV) Comprehensive MS/MS characterization of isolated myosin light chain isoforms. RA, right atrium; RV, right ventricle; LA, left atrium; LV, left ventricle; Tpm, tropomyosin; cTn, cardiac troponin; αMHC, α myosin heavy chain; βMHC, β myosin heavy chain; S1, myosin heavy chain subfragment-1.
Fig. 2
Fig. 2
Top-down MS analysis of swine RLCa. A. Zoomed-in mass spectrum showing detected RLCa species in extracts prepared from swine atrial myocardium. Triangle and oval represent RLCa associated non-covalently with sodium and potassium, respectively. B. Graph showing MS-based quantification of swine RLCa phosphorylation, which had a mean value of 0.16 ± 0.03 mol of Pi/mol of RLC (n=5). C. N-terminal fragment ions showing approximate 42.010 Da mass increase compared to the calculated masses for the corresponding un-modified fragment ions, indicating N-terminal acetylation of swine RLCa. D. Sequence table showing bond cleavages for RLCa (∼83% of all inter-residue bonds). Residues in red are amino acids not present in the previous incorrect swine RLCa sequence in the UniProtKB/Swiss-Prot database. Ovals indicate amino acids differing between swine and human RLCa. “Ac-” denotes acetylation. “p” and “pp” indicate mono- and bis-phosphorylated protein forms, respectively.
Fig. 3
Fig. 3
Localization of the phosphorylation site in swine pRLCa to Ser22 by top-down ECD MS/MS. A. Zoomed-in tandem mass spectrum showing observation of un-phosphorylated c18 ion without corresponding pc18 ion (expected position denoted by broken arrow). B. Zoomed-in tandem mass spectrum showing observation of un-phosphorylated c21 ion without corresponding pc21 ion (expected position denoted by broken arrow). C. Zoomed-in tandem mass spectrum showing observation of pc22 ion without corresponding un-phosphorylated c22 ion (expected position denoted by broken arrow). D. Zoomed-in tandem mass spectrum showing observation of pc27 ion without corresponding un-phosphorylated c27 ion (expected position denoted by broken arrow). E. Zoomed-in tandem mass spectrum showing observation of z·152 ion without corresponding pz·152 ion (expected position denoted by broken arrow). F. Zoomed-in tandem mass spectrum showing observation of pz·156 without corresponding un-phosphorylated z·156 ion (expected position denoted by broken arrow). G. Sequence table showing bond cleavages and site of phosphorylation in pRLCa. Oval indicates phosphorylated residue (Ser22) in swine pRLCa. “Ac-” denotes acetylation. “p” indicates mono-phosphorylation.
Fig. 4
Fig. 4
Top-down MS and MS/MS analyses of swine ELCa. A. Zoomed-in mass spectrum showing detected ELCa protein species in extracts prepared from the atrial myocardium of swine. Broken arrow indicates expected position of un-observed pELCa species. Star and triangle mark peaks corresponding to ELCa with NH3 loss and ELCa associated non-covalently with sodium. B. N-terminal fragment ions showing approximate 28.031 Da mass increase compared to the calculated masses for the corresponding un-modified fragment ions, indicating N-terminal di-methylation of swine ELCa. C. Sequence table for ELCa showing bond cleavages (∼95% of all inter-residue bonds). Ovals indicate amino acids differing between swine and human ELCa. “(Me)2-” denotes di-methylation. “p” indicates mono-phosphorylation.
Fig. 5
Fig. 5
Top-down MS and MS/MS analyses of swine ELCv. A. Zoomed-in mass spectrum showing swine ELCv protein species in extracts prepared from the ventricular myocardium of swine. Broken arrow indicates expected position of un-observed pELCv species. Star and triangle mark peaks corresponding to ELCv with NH3 loss and ELCv associated non-covalently with sodium, respectively. B. N-terminal fragment ions showing approximate 42.046 Da mass increase compared to the calculated masses for the corresponding un-modified fragment ions, indicating N-terminal tri-methylation of swine ELCv. C. Sequence table for ELCv showing bond cleavages (∼90% of all inter-residue bonds). Ovals indicate amino acids differing between swine and human ELCv. “(Me)3-” denotes tri-methylation. “p” indicates mono-phosphorylation.
Fig. 6
Fig. 6
Study overview showing localized mass differences in the atrial and ventricular isoforms of the ELC and RLC (left panels) from swine, as well as the modifications they represent (right panel). Highlighted is the approximate 36 mDa (0.036 Da) mass difference between tri-methylation and acetylation, which were distinguished using high-resolution top-down MS/MS in the present study.
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