Coaggregation of RNA-Binding Proteins in a Model of TDP-43 Proteinopathy with Selective RGG Motif Methylation and a Role for RRM1 Ubiquitination
Figure 1
Characterization of the aggregate proteome of HEK-293 cells overexpressing TDP-43 and aggregate prone TDP-S6.
(A) HEK-293 cells were transfected with HA-TDP-43 or HA-TDP-S6 and immunostained with anti-HA antibody for recombinant TDP-43 exclusively (red) and Hoescht stain for the nucleus (blue). Scale bars, 10 µm. (B) Western blotting (WB) of native TDP-43 in the detergent soluble (RIPA) and insoluble (urea) fractions from mock, TDP-43 and TDP-S6 transfected HEK-293 cells. 10 µg and 5 µg of detergent-soluble and insoluble sample were loaded, respectively. A ponceau S nonspecific band was used to show equal loading. (C) Workflow for quantitative proteomics using isotopic labeled internal standards. In this approach, detergent insoluble urea extracts from isotopically-labeled control HEK-293 cells are mixed equally with urea extracts prepared from mock, TDP-43 and TDP-S6 transfected cells. The samples are resolved on an SDS-PAGE gel, excised into gel slices, digested with trypsin, and analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) on a high-resolution Orbitrap mass spectrometer. The isotopically labeled peptides from the control HEK-293 cells are chemically identical to their unlabeled counterparts and serve as internal standards for the measurement of protein abundance across samples. (D) Histograms of the entire population of quantified proteins expressed as log2(light/heavy) vs. frequency for the three mixtures with Mock (null experiment), TDP-43, and TDP-S6. (E) To visualize proteins significantly changing in the TDP-43 and/or TDP-S6 models, we constructed a triple SILAC map of the differences in log2-transformed ratios. Confirming high transfection efficiency, we identified both TDP-43 and TDP-S6 as the most enriched protein component in the cell model (at the top right).