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Phosphoproteomics analysis of SKOV-3 ovarian cancer cell line after apigenin treatment

ABSTRACT: SKOV-3 ovarian cancer cell line was treated with apigenin at inhibition concentration (IC) of 10, 20 and 30. The samples were collected at 30, 60 and 90 minutes with untreated at 0 min and solvent at 90 min as control. The cells were lysed with TEAB buffer containing protease and phosphatase inhibitors. Then, disulphide bond reduction with alkylation and in solution digestion was performed with 100 Âµg of each lysate. Each condition of digested samples was labelled with one of the TMT reagents from TMT10plex with TMT11-131C reagent set. The labelled samples were pooled together and cleaned up in several steps. After that, the samples were fractionated with high Ph reverse phase system into 5 gradually increasing hydrophobicity fractions and subjected to phosphopeptide enrichment with TiO2 beads. The enriched sample was then injected into Ultimate 3000 RSLC nanosystem coupled with QExactive mass spectrometry.

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Philipp Antczak

PROVIDER: PXD054027 | JPOST Repository | Sat Jul 11 00:00:00 BST 2026

REPOSITORIES: jPOST

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			Action	DRS
	CF_080319_biorep1_tio2TMT11_fraction1.raw	Raw
	CF_080319_biorep1_tio2TMT11_fraction2.raw	Raw
	CF_080319_biorep1_tio2TMT11_fraction3.raw	Raw
	CF_080319_biorep1_tio2TMT11_fraction4.raw	Raw
	CF_080319_biorep1_tio2TMT11_fraction5.raw	Raw

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Project description:Sample preparation was conducted using an adaptation of our SimPLIT workflow10. Four cell pellets corresponding to four different colorectal cancer cell lines were lysed in a buffer containing 1% sodium deoxycholate (SDC, Merck, Cat. No. 30970), 100 mM triethylammonium bicarbonate (TEAB, Merck, Cat. No. T7408), 10% isopropanol and 50 mM NaCl, freshly supplemented with 5 mM TCEP (Thermo, Bond-breaker Cat. No 77720), 10 mM iodoacetamide (IAA, Merck, Cat. No. I1149), universal nuclease 1:2000 vol/vol (Pierce, Cat. No. 88700) and Halt protease and phosphatase inhibitor cocktail (Thermo, Cat. No. 78442, 100X) with 5 min of bath sonication. Protein concentration was measured with the Quick Start Bradford protein assay (Bio-Rad, Cat. No. 5000205). Aliquots of 30 μg of total protein from each cell line were transferred in a 96-well plate according to the 58-plex design. An E. coli protein extract (Bio-Rad, Cat. No. 1632110) was added at 1 μg or 0.5 μg across the human cell lysates. The plate was dried and the samples for the arginine-specific cleavage set (TrypR) were reconstituted in 25 μL (TMT11 subset) or 12.5 μL (TMT18 subset) of 100 mM TEAB buffer followed by the addition of 10 μL TMT11 (stock 20 μg/μL, Thermo Fischer Scientific, Cat. No. A34808) or 5 μL TMTpro18 (stock 25 μg/μL, Thermo Fischer Scientific, Cat. No. A52045), and 1h reaction at room temperature for protein-level TMT labelling. The TMT labelled samples were SpeedVac dried, and all samples (labelled and unlabelled) were reconstituted in 20 μL of 100 mM TEAB buffer. Proteins were digested overnight at room temperature by the addition of 2 μL LysC (Thermo Fischer Scientific, Cat. No. 90307) or Trypsin (Thermo Fischer Scientific, Cat. No. 90059) stock solution of 500 ng/μL in 0.1% formic acid (final enzyme 50 ng/μL, 1:30) for the LysC and TrypR sets respectively. The peptides were then fully labelled by another addition of 10 μL TMT11 or 5 μL TMTpro18. The reaction was quenched after 1h with the addition of 2 μL 5% hydroxylamine solution and premix samples were prepared by pooling 0.5 μL from each sample into 100 μL of 0.1% TFA for each sub-plex separately. In each premix, SDC was precipitated with addition of formic acid at 2% and centrifugation at 20,000 × g for 5 minutes. Supernatants were transferred into clean vials for LC-MS analysis. Following evaluation of the premix runs, samples were pooled into four sub-plexes, diluted with 100 μL of HPLC water, heated at 95 °C for 5 min, cooled down and heated for another 5 minutes. Lastly, each pool was acidified with formic acid at 2%, the precipitated SDC was removed by centrifugation and supernatants were SpeedVac dried.High-pH Reversed-phase Peptide FractionationAll four sub-plexes were pooled in 100 μL of 0.1% (v/v) ammonium hydroxide and the final peptide pool was fractionated with high pH Reversed-Phase chromatography using the XBridge C18 column (2.1 × 150 mm, 3.5 μm, Waters) on an UltiMate 3000 HPLC system over a 1% gradient in 35 min. Mobile phase A was 0.1% (v/v) ammonium hydroxide and mobile phase B was 0.1% ammonium hydroxide (v/v) in acetonitrile. Fractions were collected every 30 sec and eventually pooled into 45 samples for LC-MS analysis.

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Phosphoproteomics analysis of SKOV-3 ovarian cancer cell line after apigenin treatment

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