TY - JOUR

T1 - Rapid Affinity Purification of Tagged Plant Mitochondria (Mito-AP) for Metabolome and Proteome Analyses

AU - Niehaus, Markus

AU - Straube, Henryk

AU - Künzler, Patrick

AU - Rugen, Nils

AU - Hegermann, Jan

AU - Giavalisco, Patrick

AU - Eubel, Holger

AU - Witte, Claus Peter

AU - Herde, Marco

N1 - Funding information: 1This work was supported by the Deutsche Forschungsgemein-schaft (HE 5949/3-1 to M.H. and EU 54/4-1 to H.E.). 2Author for contact: mherde@pflern.uni-hannover.de. 3Senior author.

PY - 2020/3

Y1 - 2020/3

N2 - 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 (Arabidopsis thaliana) 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 .90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun 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 with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.

AB - 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 (Arabidopsis thaliana) 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 .90% mitochondrial proteins in the lysate. Mito-AP supports mitochondrial proteome analysis by shotgun 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 with typical mitochondrial lipids like cardiolipins and demonstrated the presence of several ubiquinones in Arabidopsis mitochondria. Affinity purification of organelles is a powerful tool for reaching higher spatial and temporal resolution for the analysis of metabolomic and proteomic dynamics within subcellular compartments. Mito-AP is small scale, rapid, economic, and potentially applicable to any organelle or to organelle subpopulations.

UR - http://www.scopus.com/inward/record.url?scp=85081146264&partnerID=8YFLogxK

U2 - 10.15488/17903

DO - 10.15488/17903

M3 - Article

C2 - 31911558

AN - SCOPUS:85081146264

VL - 182

SP - 1194

EP - 1210

JO - Plant physiology

JF - Plant physiology

SN - 0032-0889

IS - 3

ER -