Abstract
Mitochondria (mt) are double-membraned, dynamic organelles that play an essential role in a large number of cellular processes, and impairments in mt function have emerged as a causative factor for a growing number of human disorders. Given that most biological functions are driven by physical associations between proteins, the first step towards understanding mt dysfunction is to map its protein-protein interaction (PPI) network in a comprehensive and systematic fashion. While mass-spectrometry (MS) based approaches possess the high sensitivity ideal for such an endeavor, it also requires stringent biochemical purification of bait proteins to avoid detecting spurious, non-specific PPIs. Here, we outline a tagging-based affinity purification coupled with mass spectrometry (AP-MS) workflow for discovering new mt protein associations and providing novel insights into their role in mt biology and human physiology/pathology. Because AP-MS relies on the creation of proteins fused with affinity tags, we employ a versatile-affinity (VA) tag, consisting of 3× FLAG, 6 × His, and Strep III epitopes. For efficient delivery of affinity-tagged open reading frames (ORF) into mammalian cells, the VA-tag is cloned onto a specific ORF using Gateway recombinant cloning, and the resulting expression vector is stably introduced in target cells using lentiviral transduction. In this chapter, we show a functional workflow for mapping the mt interactome that includes tagging, stable transduction, selection and expansion of mammalian cell lines, mt extraction, identification of interacting protein partners by AP-MS, and lastly, computational assessment of protein complexes/PPI networks.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jiang Y, Wang X (2012) Comparative mitochondrial proteomics: perspective in human diseases. J Hematol Oncol 5:11. https://doi.org/10.1186/1756-8722-5-11
Cassarino DS, Bennett JP (1999) An evaluation of the role of mitochondria in neurodegenerative diseases: mitochondrial mutations and oxidative pathology, protective nuclear responses, and cell death in neurodegeneration. Brain Res Rev 29:1–25. https://doi.org/10.1016/S0165-0173(98)00046-0
Lin MT, Beal MF (2006) Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases. Nat Lond 443:787–795. https://doi.org/10.1038/nature05292
Ren J, Pulakat L, Whaley-Connell A, Sowers JR (2010) Mitochondrial biogenesis in the metabolic syndrome and cardiovascular disease. J Mol Med 88:993–1001. https://doi.org/10.1007/s00109-010-0663-9
Ballinger SW (2005) Mitochondrial dysfunction in cardiovascular disease. Free Radic Biol Med 38:1278–1295. https://doi.org/10.1016/j.freeradbiomed.2005.02.014
Wallace DC (2005) A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annu Rev Genet 39:359–407. https://doi.org/10.1146/annurev.genet.39.110304.095751
Floyd BJ, Wilkerson EM, Veling MT et al (2016) Mitochondrial protein interaction mapping identifies regulators of respiratory chain function. Mol Cell 63:621–632. https://doi.org/10.1016/j.molcel.2016.06.033
Malty RH, Aoki H, Kumar A et al (2017) A map of human mitochondrial protein interactions linked to neurodegeneration reveals new mechanisms of redox homeostasis and NF-κB signaling. Cell Syst 5:564–577.e12. https://doi.org/10.1016/j.cels.2017.10.010
Basso M, Giraudo S, Corpillo D et al (2004) Proteome analysis of human substantia nigra in Parkinson’s disease. Proteomics 4:3943–3952. https://doi.org/10.1002/pmic.200400848
Sokolina K, Kittanakom S, Snider J et al (2017) Systematic protein-protein interaction mapping for clinically relevant human GPCRs. Mol Syst Biol 13:918
Yao Z, Darowski K, St-Denis N et al (2017) A global analysis of the receptor tyrosine kinase-protein phosphatase interactome. Mol Cell 65:347–360. https://doi.org/10.1016/j.molcel.2016.12.004
Havugimana PC, Hart GT, Nepusz T et al (2012) A census of human soluble protein complexes. Cell 150:1068–1081. https://doi.org/10.1016/j.cell.2012.08.011
Wan C, Borgeson B, Phanse S et al (2015) Panorama of ancient metazoan macromolecular complexes. Nature 525:339–344. https://doi.org/10.1038/nature14877
Hein MY, Hubner NC, Poser I et al (2015) A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 163:712–723. https://doi.org/10.1016/j.cell.2015.09.053
Huttlin EL, Ting L, Bruckner RJ et al (2015) The BioPlex network: a systematic exploration of the human interactome. Cell 162:425–440. https://doi.org/10.1016/j.cell.2015.06.043
Krogan NJ, Cagney G, Yu H et al (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440:637–643. https://doi.org/10.1038/nature04670
Morgenstern M, Stiller SB, Lübbert P et al (2017) Definition of a high-confidence mitochondrial proteome at quantitative scale. Cell Rep 19:2836–2852. https://doi.org/10.1016/j.celrep.2017.06.014
ten Have S, Boulon S, Ahmad Y, Lamond AI (2011) Mass spectrometry-based immuno-precipitation proteomics – the user’s guide. Proteomics 11:1153–1159. https://doi.org/10.1002/pmic.201000548
Mak AB, Ni Z, Hewel JA et al (2010) A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency. Mol Cell Proteomics MCP 9:811–823. https://doi.org/10.1074/mcp.M000002-MCP201
Encinas M, Iglesias M, Liu Y et al (2000) Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells. J Neurochem 75:991–1003
Clayton DA, Shadel GS (2014) Purification of mitochondria by sucrose step density gradient centrifugation. Cold Spring Harb Protoc 2014:pdb.prot080028. https://doi.org/10.1101/pdb.prot080028
Kislinger T, Cox B, Kannan A et al (2006) Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling. Cell 125:173–186. https://doi.org/10.1016/j.cell.2006.01.044
Sowa ME, Bennett EJ, Gygi SP, Harper JW (2009) Defining the human deubiquitinating enzyme interaction landscape. Cell 138:389–403. https://doi.org/10.1016/j.cell.2009.04.042
Leung HCM, Xiang Q, Yiu SM, Chin FYL (2009) Predicting protein complexes from PPI data: a core-attachment approach. J Comput Biol J Comput Mol Cell Biol 16:133–144. https://doi.org/10.1089/cmb.2008.01TT
van Dongen S, Abreu-Goodger C (2012) Using MCL to extract clusters from networks. Methods Mol Biol Clifton NJ 804:281–295. https://doi.org/10.1007/978-1-61779-361-5_15
Westermarck J, Ivaska J, Corthals GL (2013) Identification of protein interactions involved in cellular signaling. Mol Cell Proteomics MCP 12:1752–1763. https://doi.org/10.1074/mcp.R113.027771
Sahni N, Yi S, Taipale M et al (2015) Widespread macromolecular interaction perturbations in human genetic disorders. Cell 161:647–660. https://doi.org/10.1016/j.cell.2015.04.013
Yang B, Tang S, Ma C et al (2017) Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions. Nat Commun 8:2240. https://doi.org/10.1038/s41467-017-02409-z
Vlasblom J, Jin K, Kassir S, Babu M (2014) Exploring mitochondrial system properties of neurodegenerative diseases through interactome mapping. J Proteome 100:8–24. https://doi.org/10.1016/j.jprot.2013.11.008
Hyung S-J, Ruotolo BT (2012) Integrating mass spectrometry of intact protein complexes into structural proteomics. Proteomics 12:1547–1564. https://doi.org/10.1002/pmic.201100520
Acknowledgments
We thank Babu’s group for their helpful comments. This work was funded by National Institutes of Health (R01GM106019) and Canadian Institutes of Health Research (MOP-125952; RSN-124512, 132191; FDN-154318) to MB. MB is a CIHR New Investigator (MSH-130178).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2019 Springer Nature Singapore Pte Ltd.
About this chapter
Cite this chapter
Wu, Z., Malty, R., Moutaoufik, M.T., Zhang, Q., Jessulat, M., Babu, M. (2019). A Tag-Based Affinity Purification Mass Spectrometry Workflow for Systematic Isolation of the Human Mitochondrial Protein Complexes. In: Urbani, A., Babu, M. (eds) Mitochondria in Health and in Sickness. Advances in Experimental Medicine and Biology, vol 1158. Springer, Singapore. https://doi.org/10.1007/978-981-13-8367-0_6
Download citation
DOI: https://doi.org/10.1007/978-981-13-8367-0_6
Published:
Publisher Name: Springer, Singapore
Print ISBN: 978-981-13-8366-3
Online ISBN: 978-981-13-8367-0
eBook Packages: Biomedical and Life SciencesBiomedical and Life Sciences (R0)