Representative hierarchical clustering results of mitochondrial protein profiles.

<p>Profiles from proteins with mitochondrial annotation (MITOP2 reference dataset or Gene Ontology) or mitochondrial prediction (MITOP2 SVM) were subjected to hierarchical clustering (uncentered Pearson's correlation metric, optimized leaf order, and complete linkage distance). Five representative clusters are shown that contain the five mitochondrial oxidative phosphorylation system (OXPHOS) complexes. Clusters that are referenced in the text are indicated in the HCL tree with numbers that correspond with cluster numbers defined by the MEV software.</p

LC-MS/MS data processing and protein profile generation.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g002" target="_blank"><b>Figure 2A</b></a> provides an overview for the main steps in LC-MS/MS data processing. The acquired mass spectrometry data is used to identify peptides and protein for each gel slice via database searches using the Mascot search engine. Resulting peptide identifications together with the LC-MS data are used as input for the label-free quantitation by the Ideal-Q software that integrates the chromatographic peak surfaces for each peptide from respective extracted ion currents. Quantitative information from all LC-MS/MS analyses is then used to determine the relative abundance for each peptide in every gel slice. The final step in data processing uses the peptide profiles to reconstruct the migration profile for each individual protein in the blue native gel separation. Details for the reconstruction of the protein migration profiles are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g002" target="_blank"><b>figure 2B</b></a> with data for the cytochrome <i>c</i> oxidase subunit 6 C protein. First, all peptide profiles of a protein are used to generate a similarity matrix that contains the Pearson's correlation coefficients between each peptide profile. This information is then used to calculate a similarity score for each peptide which is defined as the sum of all the Pearson's correlation coefficients for the peptide from the similarity matrix. The next step uses a Grubb's outlier test on the calculated similarity scores to discard peptide profiles that poorly correspond with the general peptide migration profile. Finally, the peptide migration profiles are ranked in descending order of their similarity score of which the 5 highest scoring peptide profiles are used to construct the protein migration profile by averaging.</p

Identified mitochondrial ribosomal complexes by hierarchical clustering.

<p>Subunits of the mitoribosome were predominantly found in 4 distinct complexes by the HCL analysis as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g005" target="_blank"><b>figure 5A</b></a>. Known subunits of the mitoribosome are bold red whereas proven interactors are in black and bold underlined. Proteins previously co-purified with mitoribosomal subunits are italic. The majority of detected MRPL subunits co-migrate in a complex of about 3 MDa in size which is referred to as the 39 S mitoribosome complex in this paper. Besides MRP subunits, six other proteins showed co-migration of which ICT1 is a proven interactor of the mitoribosome. The remaining three proteins DBT, STOML2, and CAD have previously been reported to co-purify with mitoribosomal proteins in affinity purification – mass spectrometry studies. Similar to MRPL proteins, all but one of the 28 S mitoribosomal MRPS subunits showed to co-migrate in a complex of about 1.6 MDa together with 12 other proteins that include the known mitoribosome interactor PTCD3. Four of the remaining 11 proteins have been reported to co-purify with mitoribosomal subunits in affinity purifications.This complex is referenced in the text as the 28 S mitoribosome complex.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g005" target="_blank">Figure 5A</a> also shows a smaller complex of about 300 kDa in size that includes 8 MRPS and 3 MRPL subunits of the mitoribosome together with SARM1.Finally, another complex of about 200 kDa in size was detected that appears to consist of five mitoribosomal proteins together with LRPPRC, C14ORF156 (SLIRP), and COX7A2. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g005" target="_blank"><b>Figure 5B</b></a> shows the distribution of MRP subunits versus non-MRP proteins detected in any of the four complexes and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g005" target="_blank"><b>Figure</b><i> </i><b>5C</b></a> shows the distribution of the 21 non-MRP proteins in three classes: proven interactors, proteins co-purified with mitoribosomal proteins in affinity purification – mass spectrometry studies, and proteins that have thus far not been described in literature related to the mitochondrial ribosome.</p

Co-migration of mitoribosomal proteins with previously found interactors in selected affinity purification – mass spectrometry studies.

<p>Hierarchical clustering was applied to identify co-migration of previously identified mitoribosome interactors with ribonucleotide complexes. Only gel slices of interest that correspond with the detected mitoribosomal complexes were selected for HCL analysis of each complex to increase sensitivity and specificity of the HCL analysis.</p

Detected complexes of mitoribosomal proteins together with previously identified interactors by hierarchical clustering.

<p>Migration profiles from mitochondrial ribosomal proteins and previously identified interactors were subjected to hierarchical clustering analysis to examine possible co-migration. Proteins from the mitoribosome are marked with an asterisk and functionally validated interactors are bold and underlined. Co-purified proteins from selected AP-MS studies are italic.</p

Distributions of transmembrane domains and cellular compartments of proteins in the dataset.

<p><b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g003" target="_blank"><b>Figure </b><b>3A</b></a></b> shows the tornado diagram of transmembrane domains in proteins from both the complexome profiling dataset and the complete RefSeq <i>Homo sapiens</i> database. A relative enrichment of 8% more proteins with transmembrane domains was observed for the complexome dataset with respect to the RefSeq Hs database. The tornado diagram for the cellular compartment distribution of proteins in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068340#pone-0068340-g003" target="_blank"><b>figure </b><b>3B</b></a> show an enrichment of mitochondrial proteins in our dataset compared to the RefSeq Hs database. Please note that proteins may have multiple cellular compartment GO annotations.</p