Journal of Bioanalysis & Biomedicine

Accurate Quantitation of Dystrophin Protein in Human Skeletal Muscle Using Mass Spectrometry

Abstract

Kristy J. Brown, Ramya Marathi, Alyson A. Fiorillo1, Eugene F. Ciccimaro, Seema Sharma, David S. Rowlands, Sree Rayavarapu1, Kanneboyina Nagaraju1, Eric P. Hoffman1 and Yetrib Hathout

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.