Mapping Functional Protein Neighborhoods in the Mouse Brain

Abstract

New proteomics methods make it possible to determine protein interaction maps at the proteome scale without the need for genetically encoded tags, opening up new organisms and tissue types to investigation. Current molecular and computational methods are oriented towards protein complexes that are soluble, stable, and discrete. However, the mammalian brain, among the most complicated and most heavily studied tissue types, derives many of its unique functions from protein interactions that are neither discrete nor soluble. Proteomics investigations into the global protein interaction landscape of the brain have therefore leveraged non-proteomics datasets to supplement their experiments. Here, we develop a novel, integrative proteomics pipeline and apply it to infer a global map of functional protein neighborhoods in the mouse brain without the aid of external datasets. By leveraging synaptosome enrichment and interactomics methods that target both soluble and insoluble protein fractions, we resolved protein interactions for key neural pathways, including those from refractory subcellular fractions such as the membrane and cytoskeleton. In comparison to external datasets, our observed interactions perform similarly to hand-curated synaptic protein interactions while also suggesting thousands of novel connections. We additionally employed cleavable chemical cross-linkers to detect direct binding partners and provide structural context. Our combined map suggests new protein pathways and novel mechanisms for proteins that underlie neurological diseases, including autism and epilepsy. Our results show that proteomics methods alone are sufficient to determine global interaction maps for proteins that are of broad interest to neuroscience. We anticipate that our map will be used to prioritize new research avenues and will pave the way towards future proteomics techniques that resolve protein interactions at ever greater resolution.

Cross-Linking Mass Spectrometry for Investigating Protein Conformations and Protein–Protein Interactions─A Method for All Seasons

“…So far, system-wide studies have been performed on cell lysates (human cell lines ,, and Drosophila embryos), organelles (nucleus, , mitochondria, − and synaptic vesicles , ), as well as tissues (mouse brain, plants, mouse heart), and living cells (bacterial ,,− and human cells ,,, ). System-wide experiments in which the cross-linking reaction is performed before cell lysis are in general referred to as in vivo XL-MS studies; however, a more appropriate term would be in situ XL-MS.…”

“…"Freezing" PPIs in cells allows capture of weak or transient interactions, as well as analysis of structural dynamics of proteins in their native state, including identification of physiologically relevant posttranslational modifications. 113 So far, system-wide studies have been performed on cell lysates (human cell lines 53,114,115 and Drosophila embryos 34 ), organelles (nucleus, 116,117 mitochondria, 118−121 and synaptic vesicles 122,123 ), as well as tissues (mouse brain, 124 plants, 125 mouse heart 57 ), and living cells (bacterial 55,111,126−131 and human cells 52,130,132,133 ). System-wide experiments in which the cross-linking reaction is performed before cell lysis are in general referred to as in vivo XL-MS studies; however, a more appropriate term would be in situ XL-MS.…”

Alternative proteoforms and proteoform-dependent assemblies in humans and plants

“…A set of proteins that consistently co-elute across multiple different biochemical separations can be robust evidence that those proteins interact stably. Based on this principle and the corresponding freedom from specialized reagents such as recombinant tags, antibodies, or isotope labeling, CF-MS has been broadly applied to highly diverse samples in order to discover stably interacting proteins across multiple species and kingdoms of life (Havugimana et al , 2012; Kristensen et al , 2012; Wan et al , 2015; Aryal et al , 2017; McBride et al , 2019; McWhite et al , 2020; Liebeskind et al , 2020; Pourhaghighi et al , 2020; Pang et al , 2020; Skinnider et al , 2021; Skinnider & Foster, 2021). The non-denaturing conditions that preserve protein interactions can additionally protect proteins from unfolding and non-specific degradation by cellular proteases, with degradation additionally prevented by the addition of protease inhibitors to cell lysis buffers.…”