Open AccessAccurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry
Author(s) -
Kristy J. Brown,
Ramya Marathi,
Alyson A. Fiorillo,
Eugene Ciccimaro,
Seema Sharma,
David S. Rowlands,
Sree Rayavarapu,
Kanneboyigaraju,
Eric P. Hoffman,
Yetrib Hathout
Publication year - 2013
Publication title -
journal of bioanalysis and biomedicine
Language(s) - English
Resource type - Journals
ISSN - 1948-593X
DOI - 10.4172/1948-593x.s7-001
Subject(s) - dystrophin , duchenne muscular dystrophy , mass spectrometry , biomarker , immunohistochemistry , muscular dystrophy , utrophin , tandem mass spectrometry , chemistry , medicine , computational biology , biology , pathology , chromatography , biochemistry
Quantitation of human dystrophin protein in muscle biopsies is a clinically relevant endpoint for both diagnosis and response to dystrophin-replacement therapies for dystrophinopathies. A robust and accurate assay would enable the use of dystrophin as a surrogate biomarker, particularly in exploratory Phase 2 trials. Currently available methods to quantitate dystrophin rely on immunoblot or immunohistochemistry methods that are not considered robust. Here we present a mass spectrometry based approach to accurately quantitate dystrophin protein in a total protein extract from human muscle biopsies. Our approach uses a combination of stable isotope labeled dystrophin as a spike-in standard, gel electrophoresis and high precision mass spectrometry to detect and quantitate multiple peptides of dystrophin within a complex protein mixture. The method was found highly reproducible and linear over a wide dynamic range, detecting as low as 5% of dystrophin relative to the normal amount in healthy individuals.