A high-performance liquid chromatography assay for quantification of cardiac myosin heavy chain isoform protein expression

Lemon, Douglas D.; Papst, Philip J.; Joly, Kristin M.; Plato, Craig F.; McKinsey, Timothy A.

doi:10.1016/j.ab.2010.08.041

Cited by 9 publications

(6 citation statements)

References 27 publications

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“…53 Additionally, a RPC method was developed for the separation and quantification of MHC isoforms in purified myosin. 54 Likely we could couple our SEC-based method with this RPC method for the comprehensive characterization of MHC together with isoform quantification.…”

Section: Resultsmentioning

confidence: 99%

“…Traditionally MHC isoforms can be separated by high-resolution SDS-PAGE electrophoretic methods. ,− Recently, a new method using multicolor immunofluorescence has been developed for rapid determination of MHC isoform expression, which has been applied to rat, mouse, and human skeletal muscles . Additionally, a RPC method was developed for the separation and quantification of MHC isoforms in purified myosin . Likely we could couple our SEC-based method with this RPC method for the comprehensive characterization of MHC together with isoform quantification.…”

Section: Results and Discussionmentioning

confidence: 99%

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Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry

et al. 2017

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Results and Discussionmentioning

confidence: 99%

Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry

et al. 2017

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“…Prepared PTM and TM were purified by HPLC according to the procedure of Lemon, Papst, Jol, Plato, and McKinsey () with slight modifications. The HPLC system consisted of L‐6200 Intelligent Pump and L‐6000 Pump, an L‐4000 UV Detector (Hitachi, Tokyo, Japan), and a Chromatocorder 12 (System Instruments, Tokyo, Japan) equipped with a Sepax Bio‐C18 column (4.6 × 250 mm; particle size, 3.0 μm; Sepax Technologies, Newark, DE, USA) or a Zorbax 300SB‐C18 column (4.6 × 150 mm; particle size, 3.5 μm; Agilent Technologies, Tokyo, Japan).…”

Section: Methodsmentioning

confidence: 99%

Structural differences between myofibrillar protein, paratropomyosin, and tropomyosin as revealed by high‐performance liquid chromatography

Nishio

Ushimura

Ueda

et al. 2018

Animal Science Journal

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Paratropomyosin (PTM) composes myofibril functions to weaken the rigor linkages formed between actin and myosin during postmortem aging of muscles. PTM has the similar physico-chemical properties as tropomyosin (TM) that is a regulatory protein of myofibrils. So far, it is unclear whether PTM is definitely different from TM, because the primary structure of PTM has not been determined yet. The aim of this study was to clarify structural difference of PTM from TM. PTM was prepared by column chromatography immediately after slaughter from broiler breast muscle, and purified by high-performance liquid chromatography (HPLC). Purified PTM was successfully separated from TM, and the recovered PTM molecule was reduced with dithiothreitol to separate again by HPLC. Two subunits were obtained and peptides from each digested subunit by V8 protease were recovered by HPLC, and then amino acid sequences of the peptides were analyzed by protein sequencing. As a result, some amino acid residues were replaced from that of TMα1 isoform which is the major isoform of TM, and also was different between the two subunits. Therefore, it is concluded that PTM clearly differs from TM and it is suggested that functional difference in PTM from TM is attributed to amino acid replacements in subunits composing PTM.

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“…Differences between small animal models and human cardiomyocytes are also present at the level of the myofilaments. Small rodent ventricular cardiomyocytes predominately express fast α-MHC (>94–100%) (Alpert, et al, 2002; Hamilton & Ianuzzo, 1991; Krenz, et al, 2003; Lemon, Papst, Joly, Plato, & McKinsey, 2011; J. Wang, et al, 2002) which has faster kinetics (Locher, et al, 2009; Milani-Nejad, Xu, Davis, Campbell, & Janssen, 2013; Rundell, Manaves, Martin, & de Tombe, 2005) but also higher tension cost and lower economy (Locher, et al, 2009; Rundell, et al, 2005) than the slow β-MHC (>90–95%) found in human ventricles (Miyata, Minobe, Bristow, & Leinwand, 2000; Reiser, Portman, Ning, & Schomisch Moravec, 2001).…”

Section: Small Rodent Models (Mouse and Rat)mentioning

confidence: 99%

Small and large animal models in cardiac contraction research: Advantages and disadvantages

Milani‐Nejad

Janssen

2014

Pharmacology & Therapeutics

373

310

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The mammalian heart is responsible for not only pumping blood throughout the body but also adjusting this pumping activity quickly depending upon sudden changes in the metabolic demands of the body. For the most part, the human heart is capable of performing its duties without complications; however, throughout many decades of use, at some point this system encounters problems. Research into the heart’s activities during healthy states and during adverse impacts that occur in disease states is necessary in order to strategize novel treatment options to ultimately prolong and improve patients’ lives. Animal models are an important aspect of cardiac research where a variety of cardiac processes and therapeutic targets can be studied. However, there are differences between the heart of a human being and an animal and depending on the specific animal, these differences can become more pronounced and in certain cases limiting. There is no ideal animal model available for cardiac research, the use of each animal model is accompanied with its own set of advantages and disadvantages. In this review, we will discuss these advantages and disadvantages of commonly used laboratory animals including mouse, rat, rabbit, canine, swine, and sheep. Since the goal of cardiac research is to enhance our understanding of human health and disease and help improve clinical outcomes, we will also discuss the role of human cardiac tissue in cardiac research. This review will focus on the cardiac ventricular contractile and relaxation kinetics of humans and animal models in order to illustrate these differences.

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A high-performance liquid chromatography assay for quantification of cardiac myosin heavy chain isoform protein expression

Cited by 9 publications

References 27 publications

Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry

Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry

Structural differences between myofibrillar protein, paratropomyosin, and tropomyosin as revealed by high‐performance liquid chromatography

Small and large animal models in cardiac contraction research: Advantages and disadvantages

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