Complementary proteomics strategies capture an ataxin-1 interactome in Neuro-2a cells.

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To identify protein partners of polyQ-ataxin-1 in neuronal cells under control or stress conditions, here we report our complementary proteomics strategies of proximity-dependent biotin identification (BioID) and affinity purification (via GFP-Trap pulldown) in Neuro-2a cells expressing epitope-tagged forms of ataxin-1[85Q]. These approaches allowed our enrichment of proximal proteins and interacting partners, respectively, with the subsequent protein identification performed by liquid chromatography-MS/MS. Background proteins, not dependent on the presence of the polyQ-ataxin-1 protein, were additionally defined by their endogenous biotinylation (for the BioID protocol) or by their non-specific interaction with GFP only (in the GFP-Trap protocol). All datasets were generated from biological replicates. Following the removal of the identified background proteins from the acquired protein lists, our experimental design has captured a comprehensive polyQ-ataxin-1 proximal and direct protein partners under normal and stress conditions. 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Work flow of sa", "Fig. 2. Work flow of sample preparation for mass spectrometry analysis.Following transfection to express M"], "order_basis": "paper_text_order:0", "id": "assay:1"}, {"role": "data_generation", "definition_id": "transfection", "definition_version": "99e1195c7d6f", "definition_digest": null, "required_operations": [], "support_state": "partial", "source_hashes": {"assay_lexicon": "99e1195c7d6f"}, "confidence": 0.99, "evidence": ["ion for mass spectrometry analysis.Following transfection to express MBI-ataxin-1[85Q], GFP-ataxin-1[8", "ID protocol, Neuro-2a cells were transiently transfected using Lipofectamine 2000 (ThermoFisher) to e", "trol; biotin (50 μM) was included during the transfection protocol. The untransfected control in this", "ap protocol, Neuro-2a cells were transiently transfected using Lipofectamine 2000 (ThermoFisher) to e"], "order_basis": "paper_text_order:1614", "id": "assay:2"}]}