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  A Cross-linking mass spectrometry approach defines protein interactions in yeast mitochondria

Linden, A., Deckers, M., Parfentev, I., Pflanz, R., Homberg, B., Neumann, P., et al. (2020). A Cross-linking mass spectrometry approach defines protein interactions in yeast mitochondria. Molecular and Cellular Proteomics, 19(7), 1161-1178. doi:10.1074/mcp.RA120.002028.

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 Creators:
Linden, A.1, Author           
Deckers, M., Author
Parfentev, I.1, Author           
Pflanz, R.2, Author           
Homberg, B., Author
Neumann, P., Author
Ficner, R., Author
Rehling, P.3, Author           
Urlaub, H.2, Author           
Affiliations:
1Research Group of Bioanalytical Mass Spectrometry, MPI for Biophysical Chemistry, Max Planck Society, ou_578613              
2Research Group of Bioanalytical Mass Spectrometry, MPI for biophysical chemistry, Max Planck Society, ou_578613              
3Max Planck Fellow Peter Rehling, ou_1298545              

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Free keywords: Protein cross-linking; mitochondria function or biology; quantification; modeling; yeast
 Abstract: Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins, but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labeling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labeled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly because of the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

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Language(s): eng - English
 Dates: 2020-07-012020-07-01
 Publication Status: Issued
 Pages: -
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 Rev. Type: Peer
 Identifiers: DOI: 10.1074/mcp.RA120.002028
 Degree: -

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Title: Molecular and Cellular Proteomics
Source Genre: Journal
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Pages: - Volume / Issue: 19 (7) Sequence Number: - Start / End Page: 1161 - 1178 Identifier: -