Abstract
We have used SWATH mass spectrometry to quantify 3648 proteins across 76 proteomes collected from genetically diverse BXD mouse strains in two fractions (mitochondria and total cell) from five tissues: liver, quadriceps, heart, brain, and brown adipose (BAT). Across tissues, expression covariation between genes' proteins and transcripts-measured in the same individuals-broadly aligned. Covariation was however far stronger in certain subsets than others: only 8% of transcripts in the lowest expression and variance quintile covaried with their protein, in contrast to 65% of transcripts in the highest quintiles. Key functional differences among the 3648 genes were also observed across tissues, with electron transport chain (ETC) genes particularly investigated. ETC complex proteins covary and form strong gene networks according to tissue, but their equivalent transcripts do not. Certain physiological consequences, such as the depletion of ATP synthase in BAT, are thus obscured in transcript data. Lastly, we compared the quantitative proteomic measurements between the total cell and mitochondrial fractions for the five tissues. The resulting enrichment score highlighted several hundred proteins which were strongly enriched in mitochondria, which included several dozen proteins were not reported in literature to be mitochondrially localized. Four of these candidates were selected for biochemical validation, where we found MTAP, SOAT2, and IMPDH2 to be localized inside the mitochondria, whereas ABCC6 was in the mitochondria-associated membrane. These findings demonstrate the synergies of a multi-omics approach to study complex metabolic processes, and this provides a resource for further discovery and analysis of proteoforms, modified proteins, and protein localization.
Highlights
We have used SWATH mass spectrometry to quantify 3648 proteins across 76 proteomes collected from genetically diverse BXD mouse strains in two fractions from five tissues: liver, quadriceps, heart, brain, and brown adipose (BAT)
Four of these candidates were selected for biochemical validation, where we found methylthioadenosine phosphorylase (MTAP), Sterol O-acyltransferase 2 (SOAT2), and IMPDH2 to be localized inside the mitochondria, whereas ABCC6 was in the mitochondria-associated membrane
Comprehensive Profiling of Mitochondrial Proteins Using SWATH-mass spectrometry (MS)—In this study, we generated a multi-tissue and multi-omic dataset from 8 strains of the BXD mouse population, with the primary aim of discerning how the mitochondrial proteome varies across conditions
Summary
Sample Selection—The 5 tissues studied—liver, BAT, quadriceps, brain, and heart—were collected from eight 29-week old male mice, one from each of 8 different BXD strains after overnight fasting and perfusion. Our mitochondrial isolation technique has been recently described [29], and further details are available in supplemental methods. Whole cell samples and isolated mitochondrial samples were processed the same way for protein isolation as described [30], and further details are available in supplemental. QTLs were used to perform quality control to ensure that data were broadly aligned with expectations, i.e. certain strong cis-pQTLs could be expected if the data are of high quality (detailed under the Sample Size section in supplemental methods). All datapoints were maintained in the analyses and in the supplemental datasets 2 and 3 except for comparisons between HPA and SWATH data, where protein quantifications were suppressed if the proteins had non-significant m-scores for all 8 measurements for a given tissue or fraction. For quantitative comparisons (i.e. all comparisons except tissue localization alignments in Fig. 1B and Fig. 2A–2B), e.g. the comparison of mRNA to protein level (Figs. 2E–2G), we retain all values, as the mRNA data can be used as a secondary validation of the protein quantification
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