Abstract
Human mitochondrial (mt) protein assemblies are vital for neuronal and brain function, and their alteration contributes to many human disorders, e.g., neurodegenerative diseases resulting from abnormal protein–protein interactions (PPIs). Knowledge of the composition of mt protein complexes is, however, still limited. Affinity purification mass spectrometry (MS) and proximity-dependent biotinylation MS have defined protein partners of some mt proteins, but are too technically challenging and laborious to be practical for analyzing large numbers of samples at the proteome level, e.g., for the study of neuronal or brain-specific mt assemblies, as well as altered mtPPIs on a proteome-wide scale for a disease of interest in brain regions, disease tissues or neurons derived from patients. To address this challenge, we adapted a co-fractionation–MS platform to survey native mt assemblies in adult mouse brain and in human NTERA-2 embryonal carcinoma stem cells or differentiated neuronal-like cells. The workflow consists of orthogonal separations of mt extracts isolated from chemically cross-linked samples to stabilize PPIs, data-dependent acquisition MS to identify co-eluted mt protein profiles from collected fractions and a computational scoring pipeline to predict mtPPIs, followed by network partitioning to define complexes linked to mt functions as well as those essential for neuronal and brain physiological homeostasis. We developed an R/CRAN software package, Macromolecular Assemblies from Co-elution Profiles for automated scoring of co-fractionation–MS data to define complexes from mtPPI networks. Presently, the co-fractionation–MS procedure takes 1.5–3.5 d of proteomic sample preparation, 31 d of MS data acquisition and 8.5 d of data analyses to produce meaningful biological insights.
Key points
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Mitochondrial function is achieved by protein assemblies. Studying the composition and protein–protein interactions (PPIs) of these complexes is essential for identifying and understanding human disorders resulting from abnormal PPIs. This is especially relevant for neuronal cells.
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Mitochondria are isolated and undergo chemical cross-linking to preserve the PPIs. Mitochondrial extracts are fractionated using orthogonal chromatographic methods. After proteomic mass spectrometry, an R/CRAN software package (Macromolecular Assemblies from Co-elution Profiles) is used to define mitochondrial complexes.
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Data availability
The R-package MACP is released as an open-source code under the MIT license that is available on GitHub at https://github.com/BabuLab-UofR/MACP, as well as on CRAN at https://cran.r-project.org/package=MACP. A detailed documentation on the use of MACP package in R that is provided through vignette, along with the demo data, can be downloaded from https://github.com/BabuLab-UofR/MACP/blob/main/vignettes/MACP_tutorial.Rmd. The raw co-fractionation data from this work is available at ProteomeXchange with the identifier PXD039444, in accordance with the data sharing policy. Source data are provided with this paper.
Code availability
The MACP software source code is provided on GitHub at https://github.com/BabuLab-UofR/ MACP. To guide users with MACP functionalities, illustrative datasets and output for testing, as well as documentation and tutorial vignette of MACP describing a detailed protocol on how to perform CF–MS analysis is provided on GitHub and CRAN at https://cran.r-project.org/package=MACP.
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Acknowledgements
M.T.M., M.J. and A.G. are supported by the Canadian Institutes of Health Research, Parkinson Society of Canada and/or the Saskatchewan Health Research Foundation postdoctoral fellowships. M.B. is a Chancellors Research Chair in Network Biology. This work was supported by the grants from the Canadian Institutes of Health Research (FDN-154318; PJT-186258), ALS Society of Canada, ARSACS foundation and National Institutes of Health (R01GM106019) to M.B.
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M.B. designed, conceived and supervised the study. M.Z., M.T.M. and K.B. established the iPSC procedure, as well as performed immunoblotting, immunocytochemistry, flow cytometry and mitochondrial assays. M.T.M., M.Z., A.G., M.J. and K.B. developed and optimized mt isolation and HPLC-based separations for the CF–MS procedure. H.A. performed mass spectrometry and S.P performed mass spectra searches. M.R. performed data analyses and developed MACP software, with input from M.B. M.Z., M.T.M., M.R., K.A.A. and M.B. wrote the manuscript with feedback from others. All authors read and approved the manuscript.
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Key references using this protocol
Moutaoufik, M. T. et al. iScience 19, 1114–1132 (2019): https://doi.org/10.1016/j.isci.2019.08.057
Pourhaghighi, R. et al. Cell Syst. 10, 333–350.e14 (2020): https://doi.org/10.1016/j.cels.2020.03.003
Supplementary information
Supplementary Data 1
High-quality mt protein interactions predicted in mouse brain.
Supplementary Data 2
Mt protein complexes derived from protein interactions in mouse brain.
Supplementary Data 3
Mt subunits of putative protein complexes enriched for GO annotations, human disorders and phenotypic aberrations in human disease.
Source data
Source Data Fig. 1,2,3,4,5,6,7,8
Statistical source data.
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Zilocchi, M., Rahmatbakhsh, M., Moutaoufik, M.T. et al. Co-fractionation–mass spectrometry to characterize native mitochondrial protein assemblies in mammalian neurons and brain. Nat Protoc 18, 3918–3973 (2023). https://doi.org/10.1038/s41596-023-00901-z
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DOI: https://doi.org/10.1038/s41596-023-00901-z
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