Complexome Profiling—Exploring Mitochondrial Protein Complexes in Health and Disease

Abstract

Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions. Besides, complexome profiling has been utilized to study the dynamics of OXPHOS complexes, as well as the impact of an increasing number of mutations leading to mitochondrial disorders or rearrangements of the whole mitochondrial complexome. Here, we summarize the major findings obtained by this approach; emphasize its advantages and current limitations; discuss multiple examples on how this tool could be applied to further investigate pathophysiological mechanisms and comment on the latest advances and opportunity areas to keep developing this methodology.

Uncharacterized protein c17orf80: a novel interactor of human mitochondrial nucleoids

“…Thus far we have shown that c17orf80 colocalizes with mitochondrial nucleoids and is located in close proximity to multiple nucleoid-interacting proteins. To further study these interactions and gain more evidence regarding the potential occurrence of c17orf80 in DNA-/RNA-associated protein complexes, we performed complexome profiling (CP) (Cabrera-Orefice et al, 2022; Van Strien et al, 2019; Wessels et al, 2013). CP is an unbiased approach that combines the separation of native proteins and protein complexes, typically by gel electrophoresis, with quantitative tandem MS identification of individual fractions followed by data clustering.…”

Group II Intron-Encoded Proteins (IEPs/Maturases) as Key Regulators of Nad1 Expression and Complex I Biogenesis in Land Plant Mitochondria

“…Under limited expression, or the complete absence of a specific CI subunit (or an assembly factor) the holocomplex becomes unstable, leading to the accumulation of CI-assembly intermediates of lower molecular masses, which are apparent by separation of mitochondrial complexes under native conditions (see, e.g., [ 19 ]). Based on these data, and complexome-profiling assays [ 4 , 18 , 20 ], a modular assembly pathway was proposed for CI, in which intermediates of the hydrophilic and membranous domains are sequentially and separately formed and then assembled together into the native L-shaped holo-CI enzyme [ 14 , 17 , 18 , 21 ].…”

MRPS36 provides a structural link in the eukaryotic 2-oxoglutarate dehydrogenase complex

“…Here, we explore the overall composition and architecture of mammalian OGDHC by combining complexome profiling (CP) [ 33 , 34 ] and cross-linking mass spectrometry (XL-MS) with heart mitochondria from both mice and bovine origin. Based on the XL-MS and CP data and supported by phylogenetic analyses, we propose that MRPS36 is an essential member of OGDHC, albeit exclusively in eukaryotic mitochondria.…”