Quantification of Mutant versus Wild-Type Myosin in Human Muscle Biopsies Using Nano-LC/ESI-MS

Abstract

A liquid chromatography/electrospray ionization mass spectrometry (nano-LC/ESI-MS) approach is described by which abundance of proteins (e.g., of beta-myosin heavy chain; MW 223 kDa) carrying a point mutation can be determined in tissue samples where the mutant protein is coexpressed with its wild-type forms. After enzymatic cleavage of the extracted parent protein, mutant and wild-type species of the peptide with the locus of the point mutation were quantified. Synthetic peptides, identical to wild-type and mutant peptides but labeled with stable isotopes ((13)C, (15)N), were added in known amounts as internal standards. The peak areas obtained by MS for the stable-isotope-labeled peptides and for the native peptides were used for quantification. To demonstrate the suitability of this approach we determined the relative abundance of beta-myosin with the Arg723Gly exchange in muscle biopsies of patients with Familial Hypertrophic Cardiomyopathy (HCM). For two such patients the fraction of mutated myosin was 62%, i.e., significantly different from 50%, which is quite unexpected for an autosomal dominant disease in heterozygous patients. Correlation between abundance of mutant myosin and clinical malignancy seen for several mutations in the myosin head domain emphasizes the relevance of such quantification. The approach for quantification described here is generally applicable for quantification of proteins with single point mutations even if only small amounts of tissue are available.

Unequal allelic expression of wild-type and mutated β-myosin in familial hypertrophic cardiomyopathy

“…The fraction of wild-type and mutated β-myosin was determined after myosin extraction and enzymatic cleavage using liquid chromatography/electrospray-ionization mass-spectrometry (LC/ESI-MS) or LC/MS in combination with capillary zone electrophoresis, which we previously developed [5, 26]. Figure 4 shows ESI-MS spectra of Lys-C digested native β-MHC peptides from a patient carrying mutation R723G and the corresponding synthetic stable-isotope labeled internal standard (IS) peptides.…”

“…β-MHC quantifications in M. soleus biopsies (Table 1) of two patients with mutation R723G (H27 and H28) yielded 61.6 ± 6% ( n  = 36 peptide samples) and 62.4 ± 7% ( n  = 31 peptide samples) mutated myosin of total β-MHC, respectively [5]. Preliminary β-MHC quantification of cardiac samples from patient H27 yielded essentially the same fraction of 62.7 ± 11% ( n  = 2 peptide samples) mutated myosin of total myosin as found in M. soleus.…”

“…It is unlikely that missense mutations in MYH7 cause haploinsufficiency, although relative quantification cannot fully exclude this. It has been demonstrated that β-myosin with missense mutations in the head domain is incorporated into the sarcomeres of cardiac and slow-twitch skeletal muscles [5, 21, 26]. No alterations of sarcomeric structure, packing of myofibrils or assembly of the thick filaments were found [6, 17].…”

“…The relative abundance of wild-type and mutated β-MHC was determined by liquid chromatography/electrospray-ionization mass-spectrometry as described in detail previously [5]. Quantification is based on myosin extraction and generation of peptides using endoproteinase Lys-C (Roche, Mannheim, Germany).…”

Biosensors in clinical chemistry: An overview

“…Horseradish peroxidase nanoparticles have been successfully mobilized to develop reagentless electronic biosensors for H 2 O 2 detection without calling for promoters and mediators and hence offer a great potential to develop elegant enzyme-based and competent electronic biosensors. [58]…”