ANALYTICAL CHEMISTRY, cilt.87, ss.8399-8406, 2015 (SCI İndekslerine Giren Dergi)
Actin is the major component of the cytoskeleton, playing an essential role in the structure and motility of both muscle and nonmusde cells. It is highly conserved and encoded by a multigene family. alpha-Cardiac actin (alpha CAA) and alpha-skeletal actin (alpha SKA), encoded by two different genes, are the primary actin isoforms expressed in striated muscles. The relative expression levels of alpha SKA and alpha CAA have been shown to vary between species and under pathological conditions. In particular, an increased alpha SKA expression is believed to be a programmed response of a diseased heart. Therefore, it is essential to quantify the relative expression of alpha SKA and alpha CAA, 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 a-actin extracted from muscle tissues. We thoroughly investigated all of the actin isoforms in healthy human cardiac and skeletal muscles. We found that alpha SKA is the only isoform expressed in skeletal muscle, whereas alpha CAA and alpha SKA are coexpressed in cardiac muscle. We then applied our method to quantify the a-actin isoforms in human healthy hearts and failing hearts with dilated cardiomyopathy (DCM). We found that alpha SKA 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 a-actin isoforms, enabling assessment of their clinical potential as cardiac disease markers.