https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi?db=pubmed&id=37985878&retmode=xml&tool=Litmetric&email=readroberts32@gmail.com&api_key=61f08fa0b96a73de8c900d749fcb997acc09 379858782023120720240709
1750-279918122023DecNature protocolsNat ProtocCo-fractionation-mass spectrometry to characterize native mitochondrial protein assemblies in mammalian neurons and brain.391839733918-397310.1038/s41596-023-00901-zHuman 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.© 2023. Springer Nature Limited.ZilocchiMaraM0000-0002-6138-7267Department of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.RahmatbakhshMatinehMDepartment of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.MoutaoufikMohamed TahaMTDepartment of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.BroderickKirstenKDepartment of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.GagarinovaAllaADepartment of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.Department of Biology, University of New Brunswick, Fredericton, New Brunswick, Canada.JessulatMatthewMDepartment of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.PhanseSadhnaS0000-0001-6306-0551Department of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.AokiHiroyukiHDepartment of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.AlyKhaled AKADepartment of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.BabuMohanM0000-0003-4118-6406Department of Biochemistry, University of Regina, Regina, Saskatchewan, Canada. mohan.babu@uregina.ca.engFDN-154318Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)PJT- 186258Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)Journal Article20231120
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