Quantification of mutant versus wild-type myosin in human muscle biopsies using nano-LC/ESI-MS.
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.
Becker
Edgar
E
Molecular and Cell Physiology, Medical School Hannover, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Navarro-López
Francisco
F
Francino
Antonio
A
Brenner
Bernhard
B
Kraft
Theresia
T
D001706:Biopsy; D024741:Cardiomyopathy, Hypertrophic, Familial / Q000175:diagnosis / Q000235:genetics / Q000473:pathology; D002853:Chromatography, Liquid; D006801:Humans; D009132:Muscles / Q000737:chemistry*; D009218:Myosins / Q000032:analysis* / Q000235:genetics*; D036103:Nanotechnology; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification; D017354:Point Mutation; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010446:Peptide Fragments; D009218:Myosins