The study of skeletal muscle continues to support the accurate diagnosis of mitochondrial disease and remains important in delineating molecular disease mechanisms. The heterogeneous expression of oxidative phosphorylation proteins and resulting respiratory deficiency are both characteristic findings in mitochondrial disease, hence the rigorous assessment of these at a single cell level is incredibly powerful. Currently, the number of proteins that can be assessed in individual fibres from a single section by immunohistochemistry is limited but imaging mass cytometry (IMC) enables the quantification of further, discrete proteins in individual cells. We have developed a novel workflow and bespoke analysis for applying IMC in skeletal muscle biopsies from patients with genetically-characterised mitochondrial disease, investigating the distribution of nine mitochondrial proteins in thousands of single muscle fibres. Using a semi-automated analysis pipeline, we demonstrate the accurate quantification of protein levels using IMC, providing an accurate measure of oxidative phosphorylation deficiency for complexes I–V at the single cell level. We demonstrate signatures of oxidative phosphorylation deficiency for common mtDNA variants and nuclear-encoded complex I variants and a compensatory upregulation of unaffected oxidative phosphorylation components. This technique can now be universally applied to evaluate a wide range of skeletal muscle disorders and protein targets.

骨骼肌的研究继续支持线粒体疾病的准确诊断，并且在描绘分子疾病机制方面仍然很重要。氧化磷酸化蛋白的异质表达和由此产生的呼吸缺陷都是线粒体疾病的特征性发现，因此在单细胞水平上对这些进行严格评估是非常强大的。目前，可以通过免疫组织化学从单个切片中评估单个纤维中的蛋白质数量是有限的，但成像质量流式细胞术 (IMC) 能够对单个细胞中的进一步离散蛋白质进行量化。我们开发了一种新的工作流程和定制分析，用于将 IMC 应用于具有遗传特征的线粒体疾病患者的骨骼肌活检，研究九种线粒体蛋白在数千条单根肌肉纤维中的分布。使用半自动分析管道，我们证明了使用 IMC 对蛋白质水平的准确定量，提供了在单细胞水平上对复合物 I-V 氧化磷酸化缺陷的准确测量。我们证明了常见 mtDNA 变体和核编码复合物 I 变体的氧化磷酸化缺陷的特征以及未受影响的氧化磷酸化成分的补偿性上调。这种技术现在可以普遍应用于评估广泛的骨骼肌疾病和蛋白质目标。在单细胞水平上提供复合物 I-V 氧化磷酸化缺陷的准确测量。我们证明了常见 mtDNA 变体和核编码复合物 I 变体的氧化磷酸化缺陷的特征以及未受影响的氧化磷酸化成分的补偿性上调。这种技术现在可以普遍应用于评估广泛的骨骼肌疾病和蛋白质目标。在单细胞水平上提供复合物 I-V 氧化磷酸化缺陷的准确测量。我们证明了常见 mtDNA 变体和核编码复合物 I 变体的氧化磷酸化缺陷的特征以及未受影响的氧化磷酸化成分的补偿性上调。这种技术现在可以普遍应用于评估广泛的骨骼肌疾病和蛋白质目标。