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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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			Action	DRS
	20211005_MAD_FASP_TMT_phfrac_WT_S4A_S4D_pos_neg_frac1.raw	Raw
	20211005_MAD_FASP_TMT_phfrac_WT_S4A_S4D_pos_neg_frac1_20211006153231.raw	Raw
	20211005_MAD_FASP_TMT_phfrac_WT_S4A_S4D_pos_neg_frac2.raw	Raw
	20211005_MAD_FASP_TMT_phfrac_WT_S4A_S4D_pos_neg_frac2_20211006183207.raw	Raw
	20211005_MAD_FASP_TMT_phfrac_WT_S4A_S4D_pos_neg_frac3.raw	Raw

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Analysis of Nucleolin depletion impact on the KRAS-induced proteomic changes in mouse Pancreatic ductal adenocarcinoma cells.

Project description:To reveal the role of Nuclelin (Ncl) downstream of mutant KRAS in shaping the proteome of Pancreatic Ductal Adenocarcinoma (PDAC) cells, we carried out a quantitative proteomics analysis of tumour cells isolated from an inducible mouse model of PDAC (iKras PDAC) (Ying et al., 2012). In this 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, from control or Nucleolin depleted settings. Two independent siRNA oligos were used against Ncl. All samples barcoded and pooled together using TMT 6plex labelling kit (Thermo).

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