Computational protocol: Protein kinase A can block EphA2 receptor–mediated cell repulsion by increasing EphA2 S897 phosphorylation

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Protocol publication

[…] PC3 cells were starved for 3 h and 20 min and then stimulated for 40 min with 20 μM forskolin or DMSO vehicle control. Cells were collected in ice-cold lysis buffer. Lysates were centrifuged at 13,000 × g for 10 min at 4°C to remove insoluble material and further precleared by incubation with Sepharose beads for 30 min at 4°C on a rotator. EphA2 was pulled down using biotinylated-ephrinA1-Fc ligand (BT602; R&D Systems) coupled to streptavidin beads (PI20347; Thermo Fisher Scientific). Beads were washed, and EphA2 was eluted with 6 M urea. Eluted material was treated with 10 mM DTT for 30 min at 37°C, alkylated with 40 mM indoleacetamide for 45 min at 37°C, and digested with 500 ng of trypsin (V5280; Promega, Fitchburg, WI) overnight at 37°C in an orbital shaker. Peptides were desalted using a peptide microtrap (Michrom BioResources, Auburn, CA), and phosphopeptides were enriched using a batch TiO2-based enrichment method (). Both nonenriched and enriched phosphopeptide fractions were individually separated using a MS2 HPLC connected to a 200 × 0.2–mm column, ionized using a Captive Spray Source (Michrom Bioresources), and analyzed using a decision-tree tandem mass spectrometry (MS/MS) method () in an LTQ Orbitrap Velos mass spectrometer equipped with electron transfer dissociation (Thermo Fisher Scientific). MS/MS data were searched against a concatenated target-decoy ipi.HUMAN.v.3.73 protein database (89,652 entries) using semitryptic specificity, with Sorcerer-SEQUEST on Sorcerer Enterprise (Sage-N Research, Milpitas, CA). Precursor-ion mass tolerance was ≤5.00 ppm, and product-ion mass tolerance was 0.5 atomic mass units (amu). Static carbamidomethylation of Cys (+57.0214 amu), differential oxidation of Met (+15.9949 amu), and differential phosphorylation of S, T, and Y (+79.9663 amu) were specified. Only for ETD spectra, the Versasearch script (Sage-N) specified differential modification of peptide N-termini (b- to c-ions, +17.0265 amu) and C-termini (y- to z-radical ions, −16.0187 amu) because c- and z-ions are the prominent product ions in ETD MS/MS spectra (). Filtering was with ProteinProphet (Trans-Proteomic Pipeline) at a false discovery rate of <0.01. Phosphopeptides were also filtered at the peptide level, and phosphopeptide spectra were manually inspected to verify that site-determining ions were present. Spectral quality allowed confident localization of phosphorylation sites. […]

Pipeline specifications

Software tools Comet, ProteinProphet, TPP
Application MS-based untargeted proteomics
Diseases Neoplasms, Prostatic Neoplasms
Chemicals Tyrosine