HCjE-Gi cells were cultured in 48-well plates and maintained in KSF media for up to 6 weeks. The ECM was isolated using 1% NH
4OH from cell cultures on days 1, 7, 14, 28, and 42 after seeding. The samples were dried and stored at –20°C until all of the samples were collected. The samples were then thawed, and RapiGest SF (Waters Corporation, Milford, MA, USA), an anionic surfactant, was used to solubilize the ECM directly on the dishes for subsequent in-solution tryptic digestion. RapiGest SF has been demonstrated to be highly effective for ECM protein solubilization.
26,27 First, 285 µL of 0.052% (w/v) RapiGest SF surfactant reconstituted in ammonium bicarbonate (NH
4HCO
3) (Sigma-Aldrich) was added to each sample well (final concentration 0.05%). The samples were incubated with the RapiGest SF solution for 30 minutes at RT on a plate shaker (VXR basic Vibrax, IKA Werke GmbH, Staufen, Germany) and then for 10 minutes at 80°C. The samples were cooled to RT and transferred into 1.7-mL low-binding tubes (Corning Inc., Corning, NY, USA). To ensure maximum ECM recovery, each well was rinsed with a further 100 µL of RapiGest SF, and this was transferred to the same low-binding tubes and the samples gently vortexed. Half of the protein extract (192.5 µL) was aspirated into new low-binding tubes, and 2.5 µL of 60-mM dithiothreitol (Melford Biolaboratories Ltd, Ipswich, United Kingdom) reconstituted in 25-mM NH
4HCO
3 (final concentration of 0.75 mM) was added. The samples were vortexed and incubated at 60°C for 10 minutes. Next, 2.5 µL of 180-mM iodoacetamide (Sigma) reconstituted in 25-mM NH
4HCO
3 (final concentration of 2.25 mM) was added and incubated for 30 minutes at RT in the dark. Finally, 100-ng/µL trypsin (Trypsin Gold Mass Spectrometry Grade; Promega, Fitchburg, WI, USA) was made by diluting 200-ng/µL stock solution in 50-mM acetic acid (VWR, Radnor, PA, USA) with an equal volume of 25-mM NH
4HCO
3. The samples were incubated at 37°C overnight with 2.5 µL of trypsin.