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Optimising complexome profiling for systematic exploration of DNA-/RNA-protein complexes in human mitochondria

ABSTRACT: Here we aimed to improve native gel electrophoresis-based complexome profiling (CP) method for examination of mitochondrial (mt)DNA-/RNA-protein complexes. We compared the performance of Blue Native PAGE (BNE) and high resolution Clear Native PAGE (hrCNE). We found that hrCNE much better preserves the integrity of mtDNA-/RNA-protein complexes.To further improve the detection of mtDNA-binding proteins, we treated solubilized mitochondrial samples with DNase I prior to separation on hrCNE or BNE. To characterize the detected by hrCNE mtRNA-protein complexes, we performed CP with samples treated with RNase A. To validate the utility of the method, we performed CP using mitochondria isolated from cells treated with ethidium bromide as well as from cells recovering after this treatment. Our study not only helps to validate and unveil proteins involved in mitochondrial DNA- and RNA-related processes, but also offers a convenient and systematic way for analysing these interactions that can be used equally well for the investigation of the nucleus and other DNA-/RNA-containing cell compartments.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Alisa Potter

LAB HEAD: Johannes N. Spelbrink

PROVIDER: PXD040103 | Pride | 2023-08-11

REPOSITORIES: Pride

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