Identification and Quantitation of Potential Protein Biomarkers from Patients with Muscular Dystrophy

RP-127 Duchenne Muscular Dystrophy (DMD) is a muscle disorder resulting from mutations or deletions in the dystrophin gene that results in loss of protein expression. Loss of dystrophin in muscle leads to defects in physiology and progressive muscle wasting that typically result in premature death due to cardiac dysfunction or respiratory failure. One approach to therapy in DMD involves delivery of therapeutics designed to cause skipping of the relevant mutated or deleted exon in the dystrophin gene, ultimately stimulating production of a truncated, but functional, dystrophin protein. To evaluate various therapeutic strategies like exon skipping, we attempted to identify potential biomarkers and to develop a quantitative strategy to measure and identify dystrophin protein isoforms in human skeletal muscles by MS. We used human muscle biopsy specimens to analyze dystrophin isoforms from normal human muscle and DMD muscle. Proteins from normal and DMD muscles were extracted and separated. LC/MS/MS spectra were acquired in a data-dependent mode. Proteins were searched against the human IPI Database. Data processing was done using Bioworks 3.3 for peptide ID based on Xcorr vs Charge state. The identified proteins of Normal (1013) vs DMD (865) muscle were classified by Babelomics. Their cellular component distributions were mitochondria (5.2/ 8.8%), intracellular (24.1/24.8%), membrane (14.8/11%), cytoskeleton (17.1/13.6%), cytoplasm (17.6/14.7%), nucleus (7.1/10.8%). Our results revealed the identification of proteins involved in nucleotide metabolism, Ca2+ handling, cellular stress response, key bioenegetic processes and biomarkers like dystrophin, utrophin, calpain and troponin. ICAT analysis followed by mass spectrometry detected levels of dystrophin. Improvements on the yield and recovery of dystrophy-related and clinically relevant tagged proteins are currently in progress.