Our Solutions

A Sensitive LC-HRMS Method for the Quantitation of Dystrophin in Human Muscle Tissue

Quantitation of candidate biomarkers requires specific bioanalytical assays to selectively detect and quantitate target proteins with high sensitivity in biological fluids and tissues. For Duchenne Muscular Dystrophy (DMD) therapy, dystrophin protein levels have been typically determined using immunohistochemistry and western blotting. However, recent advances in high-precision mass spectrometry analysis in combination with stable isotope spike-in strategy have made this technique well suited for the detection and quantitation of low abundant biomarkers. In this research, we present the development and validation of a mass spectrometry-based approach to accurately quantitate low levels of dystrophin protein in a total protein extract from human muscle biopsies. The approach uses a combination of stable isotope-labeled dystrophin as a spike-in standard, gel electrophoresis, and high resolution mass spectrometry.

 

Human muscle tissue extracts, spiked with stable isotope-labeled proteins, were fractionated by gel electrophoresis, bands corresponding to dystrophin were excised and digested with trypsin, and extracted peptides were analyzed by nano LC-HRMS using the ThermoFisher Q Exactive Plus. To identify dystrophin candidate surrogate peptides as well as myofiber specific muscle proteins for normalization, a Top10 data-dependent LC-MS/MS method was used. For the quantitative assessment, in the absence of commercially available standards for method development, dystrophin candidate surrogate peptides identified in the peptide mapping experiments were synthesized and used for the nano LC and PRM method optimization. Two dystrophin and two Filamin C peptides were selected to generate dystrophin protein ratios. Dilution curves ranging from 1% to 25% of normal (mix of DMD and non-DMD muscle cell extracts) showed good linearity, precision, and accuracy. The method was validated with a LLOQ of 1% of normal dystrophin levels and is currently used to support a Phase II clinical study for Duchenne Muscular Dystrophy (DMD).

THERAPEUTIC AREAS

Our deep expertise and capabilities in a broad range of therapeutic areas encompasses preclinical and early clinical studies for both small molecules and biologics. We can manage your entire program, as well as provide support research services and bioanalytical expertise.

Please see Therapeutic Areas for a fuller discussion of our capabilities and expertise.

Learn More

This site uses cookies to store information on your computer and improve your browsing experience. By remaining on this site, you consent to the placement of these cookies. Read our Cookie Policy to learn more.

Working as a flexible, attentive extension of your team, we offer all you need to move your molecule forward.