SILAC phosphoproteomics of 293-F10 cells [induced (H) vs uninduced (L)]

ABSTRACT: The objective of this experiment was to identify cellular targets of F10-mediated phosphorylation through Stable Isotope Labeling of Amino acids in Cell culture (SILAC). 293-F10 cells were propagated in heavy (H) or light (L) media for 10 cellular doublings. Mass spectrometry was used to confirm that the majority of tryptic peptides showed >90% incorporation of the heavy isotope. H-labeled cells were treated with doxycycline to induce expression of F10 for 10 hours, and then these cells were mixed with uninduced, L-labeled cells in a 1:1 ratio. Three biological replicates were obtained from separately passaged cultures and doxycycline inductions. Cells were lysed and post-nuclear supernatant proteins were precipitated and subjected to tryptic digestion. Tryptic peptides were desalted, enriched for phosphopeptides, and analyzed in triplicate by LC-MS/MS. The resulting 9 data files were pooled and phosphoprotein identification and quantification was completed using MaxQuant software (version 1.2.2.5). Differential phosphorylation in response to F10 induction was determined using an outlier score for log protein ratios. The Benjamini-Hochberg test was then used to correct for the biased statistical spread of highly abundant proteins.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

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

SUBMITTER: Michaela Pereckas  

LAB HEAD: Paula Traktman

PROVIDER: PXD005246 | Pride | 2017-02-15

REPOSITORIES: Pride