title: Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry.
abstract: Myosin heavy chain (MHC), the major component of the myosin motor molecule, plays an essential role in force production during muscle contraction. However, a comprehensive analysis of MHC proteoforms arising from sequence variations and post-translational modifications (PTMs) remains challenging due to the difficulties in purifying MHC (∼223 kDa) and achieving complete sequence coverage. Herein, we have established a strategy to effectively purify and comprehensively characterize MHC from heart tissue by combining size-exclusion chromatography (SEC) and middle-down mass spectrometry (MS). First, we have developed a MS-compatible SEC method for purifying MHC from heart tissue with high efficiency. Next, we have optimized the Glu-C, Asp-N, and trypsin limited digestion conditions for middle-down MS. Subsequently, we have applied this strategy with optimized conditions to comprehensively characterize human MHC and identified β-MHC as the predominant isoform in human left ventricular tissue. Full sequence coverage based on highly accurate mass measurements has been achieved using middle-down MS combining 1 Glu-C, 1 Asp-N, and 1 trypsin digestion. Three different PTMs: acetylation, methylation, and trimethylation were identified in human β-MHC and the corresponding sites were localized to the N-terminal Gly, Lys34, and Lys129, respectively, by electron capture dissociation (ECD). Taken together, we have demonstrated this strategy is highly efficient for purification and characterization of MHC, which can be further applied to studies of the role of MHC proteoforms in muscle-related diseases. We also envision that this integrated SEC/middle-down MS strategy can be extended for the characterization of other large proteins over 200 kDa.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Yutong
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Institutes of Biomedical Sciences, Fudan University , Shanghai, P. R. China 200433.|Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Liming
authors.initials: L
authors.lastname: Wei
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|Molecular and Cellular Pharmacology Training Program, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Wenxuan
authors.initials: W
authors.lastname: Cai
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Ziqing
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Zhijie
authors.initials: Z
authors.lastname: Wu
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Ying
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Takushi
authors.initials: T
authors.lastname: Kohmoto
authors.affiliation: Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Moss
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|Department of Cell and Regenerative Biology, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, Wisconsin 53705, United States.
authors.forename: Ying
authors.identifier: 0000-0001-5211-6812
authors.initials: Y
authors.lastname: Ge
mesh_terms: D024722:Cardiac Myosins / Q000737:chemistry* / Q000302:isolation & purification; D002850:Chromatography, Gel / Q000379:methods*; D006352:Heart Ventricles / Q000737:chemistry; D006801:Humans; D009206:Myocardium / Q000737:chemistry; D018995:Myosin Heavy Chains / Q000737:chemistry* / Q000302:isolation & purification; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C501641:MYH7 protein, human; D020033:Protein Isoforms; D024722:Cardiac Myosins; D018995:Myosin Heavy Chains