Quantitative immuno-mass spectrometry imaging of skeletal muscle dystrophin

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.

Figure 1

Dystrophin iMSI in wild type and mdx mouse quadriceps muscle. Low-resolution iMSI of whole wild-type mouse quadriceps cross section showing expected sarcolemmal distribution of dystrophin (a). High-resolution images of dystrophin iMSI in wild-type (b) and mdx mouse quadriceps (c). Quantification scale in a denotes µg kg⁻¹ gadolinium for all panels. White bar denotes 1000 µm in (a) and 50 µm in (b,c).

Figure 2

Histograms with the corresponding masks inset of the segmentation methods showing the data taken for quantification purposes using a representative sample image taken from Healthy human 1. The histograms contain the raw data (a), followed by the six different segmentation methods used, Median (b), Otsu’s (c), Sauvola (d), Phansalkar (e), k-medians clustering (f), and k-means clustering (g), respectively.

Figure 3

iMSI and histological localization of dystrophin in human muscle. iMSI for dystrophin using a Gd158-conjugated primary antibody (a,e,i,m). Dystrophin immunohistochemistry (b,f,j,n) and immunofluorescence (c,g,k,o). Laminin immunofluorescence (d,h,l,p). Healthy 1 (a–d); DMD 1 (e–h); DMD 2 (i–l); DMD 3 (m–p). Laminin immunofluorescence was used for muscle fiber localization. Quantification scales denote µg kg⁻¹ gadolinium. Scale bars denote 100 µm in all images. All immunofluorescence images were taken using Axiovision 3.0 software.