Molecular dissection of protein complexes isolated from sections of human brain

Summary Molecular studies of brain receptors and transporters have typically relied on recombinant systems, limiting insight into their organization in native tissue. Here, we develop nanobody-based immunoprecipitation coupled with native mass spectrometry to interrogate endogenous protein assemblies from post-mortem mouse and human brain sections. We exemplify our approach by characterizing the synaptic proteins VGluT1 and mGluR2. From a single mouse brain, we discover mGluR2/3 heterodimers, alongside mGluR2 homodimers. Considering regions of human brain heterodimeric mGluR2/3 is highly abundant in the OFC and sgACC (∼70% and 50%, respectively) and forms regional-specific interactions with additional synaptic proteins. In a modest cohort of biobanked human tissue, associated with depression and suicide, we find increased mGluR2/3 in the OFC. Consistent with this, mice exhibit similar associations between heterodimer levels and stress-susceptibility. Overall, our approach provides a direct means for establishing molecular-behavioural links at the level of receptor organization in brain. Competing Interest Statement T.J.E., C.A.L., J.L.B., and C.V.R. are have filed patent applications describing the use of nanobodies for native mass spectrometry, methodology for nTDMS analyses, and infrared multiphoton dissociation of native membrane proteins. J.W.M. reports payments for consulting services unrelated to the current work, including from Autobahn Therapeutics, Inc., Biohaven Pharmaceuticals, Inc., Frontier Pharma, LLC, HMP Collective, LivaNova, Plc., Merck & Co., Inc., Otsuka Pharmaceutical, Ltd, WCG Clinical, Inc., and Xenon Pharmaceuticals. C.V.R. reports unrelated consultancy services to OMass Therapeutics.