Two-Step Tag-Free Isolation of Mitochondria for Improved Protein Discovery and Quantification

Most physiological and disease processes, from central metabolism to immune response to neurodegeneration, involve mitochondria. The mitochondrial proteome is composed of more than 1,000 proteins, and the abundance of each can vary dynamically in response to external stimuli or during disease progression. Here, we describe a protocol for isolating high-quality mitochondria from primary cells and tissues. The two-step procedure comprises (1) mechanical homogenization and differential centrifugation to isolate crude mitochondria, and (2) tag-free immune capture of mitochondria to isolate pure organelles and eliminate contaminants. Mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, and enrichment yields are calculated, allowing the discovery of novel mitochondrial proteins by subtractive proteomics. Our protocol provides a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.

Automated summary

This article presents a protocol for isolating high-quality mitochondria from primary cells and tissues. The method involves mechanical homogenization and differential centrifugation to isolate crude mitochondria, followed by tag-free immune capture to obtain pure organelles and remove contaminants. The mitochondrial proteins from each purification stage are analyzed by quantitative mass spectrometry, enabling the discovery of novel mitochondrial proteins through subtractive proteomics. This protocol offers a sensitive and comprehensive approach to studying mitochondrial content in cell lines, primary cells, and tissues.