LC-MS/MS of HEK-293 inducibly expressing TDP-43 protein (WT and 3 non-functional mutants)

Aggregation of the RNA-binding protein TDP-43 is the key neuropathological feature of neurodegenerative diseases, including ALS and FTLD. TDP-43 is a ubiquitously expressed, nucleic acid-binding protein essential for human development. In physiological conditions, TDP-43 is predominantly nuclear, forms physiological oligomers and performs a wide variety of functions in the metabolism of RNA. However, whether oligomerization is required for TDP-43 functionality and whether oligomerization and RNA binding are intertwined, and if so how, remains poorly understood. To study this question, we generated three GFP-tagged human TDP-43 variants with either six point mutations that disrupt TDP-43 oligomerization (6M), five point mutations within the RNA-recognition motifs that disrupt RNA binding (RBDm) or both (6M&RBDm), and developed the corresponding four (TDP-43 WT and 3 mutants) isogenic HEK-293 cell lines expressing a single copy of GFP-TDP-43 under the control of a doxycycline-inducible promoter. Here, we induced the expression of all four TDP-43 constructs and studied the effects of the mild overexpression of the different TDP-43 variants on the human cell proteome.