Sample preparation for phosphoproteomics and proteomics analysis

Phosphoproteomics and proteomics analysis was carried out as previously described^25,29,57. AML cell pellets were lysed in 320 µL of urea buffer (8 M urea in 20 mM HEPES, pH: 8.0, supplemented with 1 mM Na₃VO₄, 1 mM NaF, 1 mM Na₄P₂O₇ and 1 mM β-glycerophosphate). AML cell lysates were homogenised by sonication for 90 cycles (30 s on 30 s off) while thawed esophagus and hepatic cell lines were homogenised for 15 cycles (30 s on & 40 s off) in Diagenode Bioruptor^® Plus and insoluble material was removed by centrifugation.

Proteins were quantified using BCA protein assay. Then, 300 μg of protein were subjected to cysteine reduction and alkylation using sequential incubation with 10 mM dithiothreitol and 16.6 mM iodoacetamide for 1 h and 30 min, respectively, at 25 °C with agitation. Trypsin beads (50% slurry of TLCK-trypsin) were equilibrated with three washes with 20 mM HEPES (pH 8.0), the urea concentration in the protein suspensions was reduced to 2 M by the addition of 900 µL of 20 mM HEPES (pH 8.0), 100 μL of equilibrated trypsin beads were added and samples were incubated overnight at 37 °C. Trypsin beads were removed by centrifugation (2000 × g at 5 °C for 5 min) and samples were divided in 250 µg for phosphoproteomics analysis and 50 µg (200 µL) for proteomics analysis.

For phosphoproteomics analysis, peptide solutions were desalted using Oasis HLB cartridges (Waters) following the manufacturer’s indications. Briefly, cartridges were set in a vacuum manifold device and the pressure was adjusted to 5 mmHg. Then, cartridges were conditioned with 1 mL acetonitrile (ACN) and equilibrated with 1.5 mL of wash solution (0.1% trifluoroacetic acid (TFA), 2% ACN). Peptides were loaded in the cartridges and washed twice with 1 mL of wash solution. Finally, peptides were eluted with 500 µL of glycolic acid buffer 1 (1 M glycolic acid, 5% TFA, 50% ACN). Enrichment of phosphorylated peptides was performed with TiO₂ Beads. Desalting eluents were normalized to 1 mL with glycolic acid buffer 2 (1 M glycolic acid, 5% TFA, 80% ACN) and incubated with 25 µl of TiO₂ buffer (500 mg TiO₂ beads in 500 µL of 1% TFA) for 5 min at room temperature. TiO₂ beads were packed by centrifugation into empty spin columns previously washed with ACN. TiO₂ beads were sequentially washed by centrifugation (1500 × g for 3 min) with 100 µL of glycolic acid buffer 2, ammonium acetate solution (100 mM ammonium acetate in 25% ACN) and three times with neutral solution (10% ACN). For phosphopeptide elution, spin tips were transferred to fresh tubes, 50 µL of elution solution 1 (5% NH₄OH, 7.5% ACN) were added and tips were centrifuged at 1500 × g for 3 min. The elution step was repeated a total of four times. Finally, samples were snap frozen, dried in a SpeedVac vacuum concentrator and phosphopeptide pellets were stored at −80 °C.

For proteomics experiments, peptide solutions were desalted using C18 + carbon top tips (Glygen Corporation). The tips were conditioned twice with 200 µL of elution solution 2 (70/30 ACN/ H2O + 0.1% TFA) and equilibrated twice with 200 µL of wash solution. Sample were loaded into the top tips and washed twice with 200 µL of wash solution. For peptide elution, the tips were transferred to fresh tubes, and peptides were eluted three times with 250 µL of elution solution 2. In all desalting steps, tips were centrifuged at 1500 x g for 5 min at 5 °C. Eluted peptide solutions were dried in a SpeedVac vacuum concentrator and peptide pellets were stored at −80 °C.