0897 Label-free Quantification of Myosin Isoforms in Porcine Skeletal Muscles

Myosin isoform (myosin heavy chain, MHC) in skeletal muscles has been usually qualified and quantified by electrophoresis, immunoblotting, and immunohistochemistry. However, it was difficult to clearly analyze myosin isoforms due to high homology among myosin isoforms. In the present study, label-free quantification (LFQ) was studied to both identify and quantify porcine myosin isoforms. Longissimus thoracis (LT), psoas major (PM), and semimembranosus (SM) muscles were taken from three pigs (180 ± 1 days old, female, 109.2 ± 2.4 kg slaughter weight) at 24 h postmortem in a slaughter house. Myofibrillar protein, which was isolated with rigor buffer (75 mM KCl, 10 mM K2HPO4, 2 mM MgCl2, 2 mM EGTA, pH 7.0), was loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis. MHC bands were cut and in-gel digested with trypsin. Spectra were obtained by analysis of liquid chromatography (LC)-mass spectrometry (MS). LFQ was performed using MaxQuant software (ver. 1.5.3.30, Max-Planck Ins., Germany). Four myosin isoforms—myosin-1 (MHC 2x), myosin-2 (MHC 2a), myosin-4 (MHC 2b), and mysoin-7 (MHC I/slow)—were identified, and their matched peptides were 193.3, 154.7, 201.2, and 77.1, respectively. Unique peptides among the matched peptides were selected for quantification of each myosin isoform. The spectral count and summed MS intensity of selected peptide were evaluated. Similar patterns were found in the relative spectral count and relative peak intensity regardless of muscle types. LT and SM muscles had a higher composition of myosin-4 than PM muscle (P < 0.05), whereas myosin-7 was higher in PM than the others (P < 0.05). Myosin-1 and myosin-2 were relatively lower than myosin-4 and myosin-7 (P < 0.05). These LFQ results showed a similar trend to previous reports, which observed the composition of myosin isoforms or myosin heavy chain-based fiber compositions in porcine skeletal muscles. Therefore, LFQ can be a useful approach to overcome the problem of myosin quantification caused by high homology among their isoforms.