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. 2020 Oct 19;10:17599. doi: 10.1038/s41598-020-74718-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Quantification of percent mitochondrial enrichment using subcellular proteomics. (A) Heatmap displaying abundance (Log2) of mitochondrial and non-mitochondrial high confidence proteins from isolated mitochondria samples prepared from BAT, HRT, KID, and LIV. Mitochondrial proteins are indicated in the legend by a ‘red’ mark. Mitochondrial proteins were assigned using the MitoCarta 2.0 database. (B) Summed total abundance of all proteins, relative to mitochondrial proteins. (C) Mitochondrial enrichment factors per tissue, derived from the ratio of mitochondrial to total protein abundance. (D) Citrate synthase (CS) activity in isolated mitochondria, normalized to protein. (E) CS abundance. (F–J) Correlation between CS activity and MitoCarta enrichment. (J) Overlapping proteins per tissue that correlate with MitoCarta enrichment. (K) CS activity normalized to protein and corrected for each sample’s mitochondrial enrichment factor (MEF). To do this, CS activity displayed in panel (D) was multiplied by the sample-specific MEF. (A) Figure generated using R Studio version 3.6.2 (heatmap.plus). (B–I, K) Figures were generated using GraphPad Prism 8 software (Version 8.4.2) (J) Figure generated using https://bioinformatics.psb.ugent.be/webtools/Venn/. Data information: Data are presented as mean ± SEM, N = 5/group. **P < 0.01, ***P < 0.001, ****P < 0.0001, ^#P < 0.05 (Student’s t-test).