1cc oasis cartridges

Phosphoproteomic was performed as described previously ⁵² with some modifications ⁵³ . Three 10cm dishes of IMR90 ER:RAS cells were seeded (2 × 10⁶ per plate) per condition. 7 days after 4OHT induction, cells were lysed in urea lysis buffer (8 M Urea, 20 mM HEPES) containing phosphatase inhibitors and, after diluting to <2M Urea, 400 μg proteins were digested using immobilized trypsin overnight at 37°C. The resulting peptides were desalted by RP-SPE in 1cc Oasis cartridges (Waters Corp.) using manufacturer’s instructions with the exception that peptide elution was with 1 mL of 2M glycolic acid, 50% acetonitrile, 5% trifluroracetic acid. Eluted peptides were spiked with 50 μL TiO₂ (50% slurry) that was then collected by centrifugation and washed with washing solution (2 M glycolic acid, 50% acetonitrile, 5% trifluoroacetic acid) three times. Phosphopeptides were eluted from TiO₂ using ammonium hydroxide and dried in a speed vacuum centrifuge until the time of analysis. Samples were solubilised and separated by nano-LC as per the proteomic analyses, but a top 10 MSA (multi-stage activation) method was utilized for increased coverage for neutral loss phosphoproteomic data acquisition.