Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

pmic7284-sup-0001-FigureS1.tif1182KFigure S1. Differential centrifugation of the 600 x g supernatant on a Percoll™ gradient at 50,000 x g for 90 min. Due to their higher density (1.09–1.11 g/ml) the mitochondria sediment more quickly, can thus be separated from the lysosomal fraction (1.04–1.07 g/ml) and be aspirated from the ring close to the bottom of the centrifugation tube.
pmic7284-sup-0002-FigureS2.tif1245KFigure S2. Electron micrographs (x 4,646) of the purified mitochondrial fraction from the five different organs after density gradient centrifugation. The varying morphologies and sizes of the mitochondria from the different tissues can clearly be seen.
pmic7284-sup-0003-FigureS3.tif3040KFigures S3. A-D: Comparison of 2DE gel sections of all five organs for all deviating protein spots from Table 1. If several spots were identified on the same gel segment, they were depicted by different line styles that are repeated on the left. Below the name of the protein and the respective spot number the molecular weight and the pI (MW[pI]) were provided.
pmic7284-sup-0004-FigureS4.tif666KFigures S4. A-E: Depiction of the differentially expressed protein spots in the higher molecular weight range above 50 kDa in all five organs. The spots are marked on the 2DE-gel with specific colors and line-styles that are repeated on the left.
pmic7284-sup-0005-FigureS5.tif628KFigures S5. Reproducibility of quantitative data in 2DE patterns from mitochondrial preparations of all five organs. (Left column) Relative spot volumes [%] from two technical replicates depicted by X-Y plot. The red line depicts the regression line and R2 is the Pearson product moment coefficient. (Right column) Quantile-Quantile (Q-Q) plot of the replicates. Significant deviations from the 45o line are only seen for all samples below a relative spot volume of 0.01%. These spots were subsequently excluded from quantitative analysis.
pmic7284-sup-0006-FigureS6.tif5661KFigure S6. (A) Comparison of the same protein in 5 different tissues. Enlarged sections of 2DE-gels of the five tissues visualized as 3D-landscapes through the ProteomeWeaver™ software (Definiens, Munich, Germany) and as plain view. Protein spot 4 (∆) is present with varying spot volumes in brain (3.80); skeletal muscle (4.81), kidney (8.41) and heart muscle (8.98) and absent in liver. This protein spot was identified as the mitochondrial Succinyl-CoA:3-ketoacid-CoA transferase 1 (SCOT_MOUSE). Spots in the vicinity of spot 4 were identified as (1) mitochondrial Delta-1-pyrroline-5-carboxylate dehydrogenase (AL4A1_MOUSE) and (2) mitochondrial Aldehyde dehydrogenase (ALDH2_MOUSE) (B) Variation of a single spot between 10 biological replicates. Variation of spot 4 (∆) within the proteome of skeletal muscle in 10 individual mice (biological replicates). The 3D view illustrates the variation of the individual densities between 2.59 and 7.13 (4.81±1.28).
pmic7284-sup-0007-FigureS7.tif7629KFigure S7. Mitochondrial localization of the proteins that have been newly discovered within the mitochondrial proteome. Plasmids encoding the respective C-terminally GFP-tagged full-length Akr1a1-, Cryz-, Cat-, and Ppia-proteins were co-transfected into COS1 cells with a mitochondrial localization plasmid (pTagRFP-mito).
pmic7284-sup-0008-FigureS8.tif254KFigure S8. The success rate for indentification of protein spots through excision from the 2DE gel and subsequent ESI-MS/MS analysis crucially depends on the relative spot volume. 500 spots have been analyzed in total and for relative spot volumes below 0.05% the identification rate fell below 30%.
pmic7284-sup-0009-FigureS9.tif948KFigure S9. A-D: High-resolution scans of the preparative 2DE gels from mouse brain, heart, skeletal muscle, liver, and kidney. All spots listed in Table 1 are highlighted and labeled with the respective spot numbers. Spots depicted in red color are also highlighted on Supplementary Figure 4A-D, where the running time of the second dimension gel was extended by 50%, which improved the resolution of high molecular weight proteins.
pmic7284-sup-0010-FigureS10.tif1213KFigure S10
pmic7284-sup-0011-FigureS11.tif1275KFigure S11
pmic7284-sup-0012-FigureS12.tif1447KFigure S12
pmic7284-sup-0013-FigureS13.tif1333KFigure S13
pmic7284-sup-0014-FigureS14.tif3322KFigure S14
pmic7284-sup-0015-FigureS15.tif3317KFigure S15
pmic7284-sup-0016-FigureS16.tif2003KFigure S16
pmic7284-sup-0017-FigureS17.tif624KFigure S17
pmic7284-sup-0018-FigureS18.tif601KFigure S18
pmic7284-sup-0019-FigureS19.tif619KFigure S19
pmic7284-sup-0020-FigureS20.tif586KFigure S20
pmic7284-sup-0021-TableS1.xls69KTable S1. List of the identified proteins that differ between the five mouse tissues sorted by the gene name. Predicted MW and pI were calculated – if known – with the mitochondrial import sequence removed. Several classes of identification could be separated: “ø” complete absence in the 2DE gel; “position” the spot was identified by its position with regard to other known anchor spots in the gels of other organs. “number” the number depicts the MOWSE score (number of matched peptides) and thus the certainty of the spot identification. Spot numbers marked by an asterisk were determined in the high molecular range gels. If a protein was associated with an inherited human disease, the OMIM number and the name of the disease are given in the last two columns of the table.
pmic7284-sup-0022-TableS2.xls160KTable S2. The table depicts the sequences and identification scores of all fragment ions from all identified proteins.
pmic7284-sup-0023-S1.doc1037KZIP-Compressed original fragment ion files:

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