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Proteomic analyses reveal that loss of TDP-43 affects RNA processing and intracellular transport
Transactive response DNA-binding protein 43 (TDP-43) is a predominantly nuclear, ubiquitously expressed RNA and DNA-binding protein. It recognises and binds to UG repeats and is involved in pre-mRNA splicing, mRNA stability and microRNA metabolism. TDP-43 is essential in early embryonic development but accumulates in cytoplasmic aggregates in amyotrophic lateral sclerosis (ALS) and tau-negative frontotemporal lobar degeneration (FTLD). It is not known yet whether cytoplasmic aggregates of TDP-43 are toxic or protective but they are often associated with a loss of TDP-43 from the nucleus and neurodegeneration may be caused by a loss of normal TDP-43 function or a gain of toxic function. Here we present a proteomic study to analyse the effect of loss of TDP-43 on the proteome. Our results indicate that TDP-43 is an important regulator of RNA metabolism and intracellular transport. We show that Ran-binding protein 1 (RanBP1), DNA methyltransferase 3 alpha (Dnmt3a) and chromogranin B (CgB) are downregulated upon TDP-43 knockdown. Subsequently, transportin 1 level is increased as a result of RanBP1 depletion. Improper regulation of these proteins and the subsequent disruption of cellular processes may play a role in the pathogenesis of the TDP-43 proteinopathies ALS and FTLD.
Sample Processing Protocol
Nuclear and cytosolic fractions from control and TDP-43 knockdown cells (n=3) were separated by 4-12% Bis-Tris gel and silver stained. Each lane was cut into 8 gel pieces and digested with trypsin using an Investigator ProGest (Digilab) robotic digestion system. Tryptic peptides were separated on a nanoflow LC system (Dionex Ultimate 3000, Thermo Fisher Scientific) using reversed phase columns (PepMap C18, 25cm x 75um) and eluted with a 40min gradient (10-25% B in 35 min, 25-40% B in 5min, 90% B in 10min, and 2% B in 20min, where A is 2% ACN, 0.1% formic acid in HPLC H2O and B is 80% ACN, 0.1% formic acid in HPLC H2O). Sequentially eluted peptides were directly analysed by tandem mass spectrometry (LTQ Orbitrap XL, Thermo Fisher Scientific) using full ion scan mode over the mass-to-charge (m/z) range 400-1600. Tandem MS (MS/MS) was performed on the top 6 ions using data-dependent mode with dynamic exclusion.
Data Processing Protocol
Peak lists were generated by extract_msn_com.exe (version 5.0, Thermo Fisher Scientific) and searched against UniProt/SwissProt human database (version 2014_01, 20267 protein entries) using Mascot (version 2.3.01, Matrix Science). The mass tolerance was set at 10ppm for the precursor ions and at 0.8 Da for fragment ions. Carboxyamidomethylation of cysteine was used as a fixed modification and oxidation of methionine as a variable modification. Two missed cleavages were allowed. Search results were loaded into Scaffold (version 4.3.2) using MudPIT, LFDR scoring, and protein cluster analysis. The following filters were used: peptides probability > 95%, protein probability >99%, minimum 2 unique peptides identified. Normalized spectra counts were used for quantification.
Štalekar M, Yin X, Rebolj K, Darovic S, Troakes C, Mayr M, Shaw CE, Rogelj B. Proteomic analyses reveal that loss of TDP-43 affects RNA processing and intracellular transport. Neuroscience. 2015 May 7;293:157-70 PubMed: 25743254
|#||Accession||Title||Proteins||Peptides||Unique Peptides||Spectra||Identified Spectra||View in Reactome|
|1||43311||Effect of the TDP-43 loss of function on the proteome - Control3 Nuclear band 8||1||6||4||413||6||
|2||43255||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 1||364||1125||625||2082||995||
|3||43312||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 2||380||1259||697||2110||1105||
|4||43254||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 3||304||1367||665||2152||1071||
|5||43317||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 4||312||1228||611||2090||954||
|6||43318||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 5||270||1422||650||2163||1021||
|7||43315||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 6||269||1233||682||2154||1084||
|8||43316||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 7||236||1284||816||2181||1198||
|9||43296||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Cytosolic band 8||185||1127||686||1974||1042||
|10||43297||Effect of the TDP-43 loss of function on the proteome - Knockdown1 Nuclear 1||228||773||372||1852||610||