A Cross-linking Mass Spectrometry Approach Defines Protein Interactions in Yeast Mitochondria

Abstract

<jats:p>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 <jats:italic>Saccharomyces cerevisiae</jats:italic> 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, <jats:italic>i.e.</jats:italic> 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 <jats:italic>c</jats:italic> oxidase. BN-PAGE analyses of <jats:italic>min8</jats:italic>Δ mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome <jats:italic>c</jats:italic> oxidase.</jats:p>

Defining the architecture of the human TIM22 complex by chemical crosslinking

“…The water soluble, amine‐amine bifunctional crosslinker, bis(sulfosuccinimidyl)suberate (BS3), has previously been successfully used for the XL‐MS analysis of mitochondrial proteins [53]. BS3 has an arm length of 11.4 Å, providing a medium length range for crosslinking of TIM22 components.…”

Towards low false discovery rate estimation for protein-protein interactions detected by chemical cross-linking

“…A much chosen subject of study concerns PPIs in isolated mitochondria represented in Table 1 by five recent examples [13][14][15][16][17]. There is partial overlap between the PPIs identified in these studies, but the fraction of identified inter-protein cross-linked peptides varied from about 3% to about 60%.…”

“…Furthermore, FDR can be expressed at different levels, varying from the cross-linked spectrum (CSM) level down to the PPI level (section 4) [10,15,20]. The different levels used to express the FDR in similar types of CXMS studies like the mitochondrial interactome examples [13][14][15][16][17] has been confusing. It underscores the importance of the summons of the CXMS community for the development of standards for reliable FDR estimation for inter-protein peptide pairs [6].…”

“…Linden e.a. [16] DSS [24] non-cleavable -Saccharomyces cerevisiae isolated mitochondria Linden e.a. [16] Homo sapiens isolated mitochondria Ryl e.a.…”

“…[16] DSS [24] non-cleavable -Saccharomyces cerevisiae isolated mitochondria Linden e.a. [16] Homo sapiens isolated mitochondria Ryl e.a. [15] a These papers both describe how potential false positive inter-protein cross-links can be recognized in a dataset subjected to a target-decoy approach and show development of effective filters to lower the FDR for inter-protein peptide pairs.…”