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 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.

Comprehensive characterization of monoclonal antibody by Fourier transform ion cyclotron resonance mass spectrometry

“…27 Compared with the bottom-up approach, middle-down MS reduces the chance of proteoform information loss by analyzing larger polypeptides (> 3 kDa) that may contain multiple PTMs and unique isoform sequences. [28][29][30] Moreover, as the typical bond cleavage of mAbs in a middle-down experiment is 50%~70%, this middle-down approach improves the bond cleavage coverage in protein characterization that is limited in top-down MS. 25,26,31 Although a variety of MS approaches have been established for the characterization of therapeutic mAbs, 32 most of them focused on the high-abundance N-linked glycoforms. 33,34 High mass accuracy from isotopically resolved high-resolution MS data could be used to increase the confidence level of protein identification with detection of PTMs and variations in sequence.…”

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

“…64,67,68,77,79,85,91,139 Additionally, a few MD studies have been carried out using the hybrid LTQ-FT-ICR-MS also. 75,84,185,186…”

“…186 Other soware, such as Pro-sightPC and MASH-Suite, which were primarily developed for analysis of TD proteomic data, have also been applied well for the purpose of analyzing MD proteomic data. 66,67,70,88,89,99,172,185 Algorithms to process the raw data of MDP for the purpose of deconvolution are, Xtract, THRASH 249 and MS-Deconv. 75,91,257 Among these, Xtract and THRASH algorithms have been used mostly to analyze FT-MS data of longer proteolytic peptides.…”

Post-translational modification patterns on β-myosin heavy chain are altered in ischemic and non-ischemic human hearts

“…In a study by Kawai et al examining PTMs on MYH6, the predominant murine myosin isoform, 31 phosphorylation sites were identified in control hearts with a number of these residues not phosphorylated in HCM hearts (21). In addition, Jin et al identified acetylated, methylated, and trimethylated residues in human β-MHC using size-exclusion chromatography (SEC)/middle-down mass spectrometry (MS) (22).…”