(1) Label your cells with SILAC (at least 5 population doublings to ensure complete incorporation of isotope-labeled amino acids).
(2) Serum-starve your cells before pumping for approximately 12 h.
(3) Connect pumps with tubing as outlined in Figure 1: pump P1 and P2 run with the same speed (in our case 800 µl/min), P1 delivers medium containing the stimulus, P2 delivers cells (in suspension or growing on microcarrier beads). Pump P3 delivers the quenching reagent, in our case 80% ethanol prechilled to -20°C and further cooled by -20°C cooling aggregates. To reach proper quenching, pump P3 should run approximately 5 to 10 times faster than pumps P1 and P2 (in our case 8 ml/min). It is possible to place pumps P1, P2, and the stimulation tube in an incubator to perform experiments at 37°C.
(4) Adjust the tube length determining the time of stimulation according to your pumping speed (speed of pump P1 + speed of pump P2) and the tubing diameter to reach the desired stimulation time: volume (V) = π × r2 × length (L). We used 1 mm i.d. Teflon tubes with the following lengths:(a) 1 s: 3.4 cm, (b) 5 s: 17 cm, (c) 10 s: 34 cm, and (d) 60 s: 204 cm.
(5) Prepare cell suspension and double concentrated stimulation medium. We used 1.5-4×108 cells per time point resuspended in 5 or 10 ml of medium and medium containing 300 ng/ml EGF (150 ng/ml final concentration).
(6) Pump sample through the system.
(7) Pellet your cells by centrifugation.
(8) Discard supernatant, wash pellet with ice-cold PBS, and pellet again.
(9) Lyse cells with lysing buffer containing protease and phosphatase inhibitors.
(10) Immunoprecipitate your complexes of interest, separate them by SDS-PAGE and digest them enzymatically, e.g. with trypsin. We used preconjugated 4G10 antibody to precipitate phosphotyrosine containing proteins.
(11) Analyze resulting peptide mixture by LC-MS. In the case of phosphopeptide analysis, phosphopeptides should be enriched prior LC-MS analysis, e.g. by TiO2.
(12) Analyse your data and quantitate peptides of interest, e.g. by MSQuant software.