title: Effective top-down LC/MS+ method for assessing actin isoforms as a potential cardiac disease marker.
abstract: Actin is the major component of the cytoskeleton, playing an essential role in the structure and motility of both muscle and nonmuscle cells. It is highly conserved and encoded by a multigene family. α-Cardiac actin (αCAA) and α-skeletal actin (αSKA), encoded by two different genes, are the primary actin isoforms expressed in striated muscles. The relative expression levels of αSKA and αCAA have been shown to vary between species and under pathological conditions. In particular, an increased αSKA expression is believed to be a programmed response of a diseased heart. Therefore, it is essential to quantify the relative expression of αSKA and αCAA, which remains challenging due to the high degree of sequence similarity between these isoforms (98.9%). Herein, we developed a top-down liquid chromatography/mass spectrometry-based ("LC/MS+") method for the rapid purification and comprehensive analysis of α-actin extracted from muscle tissues. We thoroughly investigated all of the actin isoforms in healthy human cardiac and skeletal muscles. We found that αSKA is the only isoform expressed in skeletal muscle, whereas αCAA and αSKA are coexpressed in cardiac muscle. We then applied our method to quantify the α-actin isoforms in human healthy hearts and failing hearts with dilated cardiomyopathy (DCM). We found that αSKA is augmented in DCM compared with healthy controls, 43.1 ± 0.9% versus 23.7 ± 1.7%, respectively. As demonstrated, top-down LC/MS+ provides an effective and comprehensive method for the purification, quantification, and characterization of α-actin isoforms, enabling assessment of their clinical potential as cardiac disease markers.
authors.affiliation: Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Department of Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Yi-Chen
authors.initials: YC
authors.lastname: Chen
authors.affiliation: Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Serife
authors.initials: S
authors.lastname: Ayaz-Guner
authors.affiliation: Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Peng
authors.affiliation: Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Lane
authors.affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Locher
authors.affiliation: Department of Surgery, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Takushi
authors.initials: T
authors.lastname: Kohmoto
authors.affiliation: Department of Physiology and Pharmacology, Department of Clinical Neuroscience, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Lars
authors.initials: L
authors.lastname: Larsson
authors.affiliation: Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Moss
authors.affiliation: Department of Cell and Regenerative Biology, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Department of Chemistry, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.|Human Proteomics Program, School of Medicine and Public Health, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Ying
authors.initials: Y
authors.lastname: Ge
mesh_terms: D000199:Actins / Q000097:blood* / Q000737:chemistry; D015415:Biomarkers / Q000097:blood*; D002853:Chromatography, Liquid; D006331:Heart Diseases / Q000097:blood*; D006801:Humans; D013058:Mass Spectrometry; D009206:Myocardium / Q000737:chemistry / Q000473:pathology; D020033:Protein Isoforms / Q000097:blood*; D012015:Reference Standards; D013997:Time Factors
chemical_list: D000199:Actins; D015415:Biomarkers; D020033:Protein Isoforms