Rapid affinity purification of tagged plant mitochondria (Mito-AP) for metabolome and proteome analyses

The isolation of organelles facilitates the focused analysis of subcellular protein and metabolite pools. Here we present a technique for the affinity purification of plant mitochondria (Mito-AP). The stable ectopic expression of a mitochondrial outer membrane protein fused to a GFP:Strep-tag in Arabidopsis exclusively decorates mitochondria, enabling their selective affinity purification using magnetic beads coated with Strep-Tactin. With Mito-AP, intact mitochondria from 0.5 g plant material were highly enriched in 30-60 min, considerably faster than with conventional gradient centrifugation. Combining gradient centrifugation and Mito-AP techniques resulted in high purity of over 90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shot-gun proteomics. The relative abundances of proteins from distinct mitochondrial isolation methods were correlated. A cluster of 619 proteins was consistently enriched by all methods - among these were several proteins that lack subcellular localization data or that are currently assigned to other compartments. Mito-AP is also compatible with mitochondrial metabolome analysis by triple-quadrupole and orbitrap mass spectrometry. Mito-AP preparations showed a strong enrichment for typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinons in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool to reach higher spatial and temporal resolution for the analysis of the metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle sub-populations.