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Analysis of the impact of Nucleolin phospho-mutants on the proteome of pancreatic ductal adenocarcinoma cells.

ABSTRACT: To reveal the impact of Nucleolin (Ncl) phosphorylation on oncogenic KRAS-induced changes in the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a quantitative proteomic analysis on tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012), which were ectopically expressing wild-type or different phospho-mutants of Ncl. In this mouse model, oncogenic Kras (G12D) expression can be controlled by administration of doxycycline (Dox). Tandem Mass Tagging (TMT) was employed for quantitative analysis of Dox induced (24hrs) vs. non-induced iKras-PDAC cells, which were privously transfected with three different expression constructs of mouse myc-tagged Ncl. These constructs encoded for wild-type Ncl (WT Ncl), a phospho-defective mutant with four Serine to Alanine mutations on S28, S34, S41, and S42 sites (S4A Ncl), and a phospho-mimicking mutant with the same Serines mutated to Aspartic Acid (S4D Ncl). Cells were subjected to whole cell lysis, followed by Trypsin digestion, TMT barcoding with TMT 6plex kit (Thermo), and peptide fractionation, before analysis by LC-MS/MS.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

DISEASE(S): Carcinoma

SUBMITTER: Faraz Mardakheh

LAB HEAD: Faraz K. Mardakheh

PROVIDER: PXD038496 | Pride | 2023-02-12

REPOSITORIES: Pride

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