Quantitative immuno-mass spectrometry imaging of skeletal muscle dystrophin

David P Bishop,Thomas Lockwood,Jonathan Wanagat,Florian Barthelemy,M Carrie Miceli,Philip A Doble,Stanley F Nelson,Nerida Cole,Elizabeth M Gibbs,Mika T Westerhausen,Rachelle H Crosbie

doi:10.1038/s41598-020-80495-8

Abstract

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. Current approaches have poor precision, require large amounts of tissue, and are difficult to standardize. This paper presents an immuno-mass spectrometry imaging method using gadolinium (Gd)-labeled anti-dystrophin antibodies and laser ablation-inductively coupled plasma-mass spectrometry to simultaneously quantify and localize dystrophin in muscle sections. Gd is quantified as a proxy for the relative expression of dystrophin and was validated in murine and human skeletal muscle sections following k-means clustering segmentation, before application to DMD patients with different gene mutations where dystrophin expression was measured up to 100 µg kg−1 Gd. These results demonstrate that immuno-mass spectrometry imaging is a viable approach for pre-clinical to clinical research in DMD. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation.

Highlights

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin
The fiber-specific dystrophin distribution is clearly seen for the WT mouse, whereas little to no dystrophin expression was observed in the mdx mouse model with no revertant fibers identified within the region of interest
Up to 62% of subjects demonstrated fibers with residual or trace dystrophin[13]. Those with nonsense mutations are predicted to be “typical” DMD patients based on history and the age at loss of ambulation[33,34,35,36]

Summary

Introduction

Emerging and promising therapeutic interventions for Duchenne muscular dystrophy (DMD) are confounded by the challenges of quantifying dystrophin. It rapidly quantified relative dystrophin in single tissue sections, efficiently used valuable patient resources, and may provide information on drug efficacy for clinical translation. Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine and College of Letters and Sciences, University of California, Los Angeles, Los Angeles, CA, USA. Typically have less than 3% of normal levels[14], and mice models show that 15% of normal expression is sufficient to provide significant b enefits[11]

Methods

Results

Conclusion