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 non-muscle 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 co-expressed 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 23.6 ± 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.

Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics

“…Our group has previously studied Tpm isoforms in swine (Peng et al 2013a) and human (Peng et al 2013b) cardiac muscles by top-down MS, using an offline multi-step purification strategy. Recently, we have developed a simple and robust top-down liquid chromatography (LC)/MS+ (Thangaraj et al 2010; Whitelegge et al 2002) strategy for the analysis of myofilament proteins extracted from cardiac tissues (Chen et al 2015; Peng et al 2014), which greatly simplified the purification procedure of myofilament proteins. Herein, we have employed this strategy to characterize Tpm isoforms present in different types of skeletal muscle from three species (swine, rat, and human) toward a better understanding of the functional role of Tpm in skeletal muscles.…”

Top-down Proteomics: Technology Advancements and Applications to Heart Diseases

“…However, precise quantification of these two isoforms is difficult because the two isoforms differ from each other by only 32 Da due to two juxtaposed amino acids [98]. Although gene expression levels are typically quantified by measuring the levels of the protein-specific mRNAs, studies have suggested that protein levels do not necessarily correlate with mRNA levels [99].…”

“…Although gene expression levels are typically quantified by measuring the levels of the protein-specific mRNAs, studies have suggested that protein levels do not necessarily correlate with mRNA levels [99]. By employing high-resolution top-down MS, Chen et al were able to distinguish between α-cardiac and α-skeletal actin in both human and swine heart tissue [98]. Moreover, top-down proteomics revealed that there is a marked upregulation of α-skeletal actin relative to α-cardiac actin in the failing hearts as compared to the donor hearts, which can potentially serve as a biomarker for cardiac dysfunction [98].…”

“…By employing high-resolution top-down MS, Chen et al were able to distinguish between α-cardiac and α-skeletal actin in both human and swine heart tissue [98]. Moreover, top-down proteomics revealed that there is a marked upregulation of α-skeletal actin relative to α-cardiac actin in the failing hearts as compared to the donor hearts, which can potentially serve as a biomarker for cardiac dysfunction [98]. …”

Top-down proteomics: applications, recent developments and perspectives

“…A rapid purification, reliable quantification, and comprehensive characterization of α-actin isoforms due to genetic variations together with PTMs was recently developed [144]. It was reported that using top-down MALDI-FTICR-MS platform, the mass analysis of intact human serum peptides and small proteins with isotopic resolution up to ≈15 kDa and identified new proteoforms from an accurate measurement of mass distances [145].…”