RP-HPLC and MS Analyses

The apical and basolateral compartments were evaluated in RP-HPLC using a column Onyx Monolithic C18 100 mm × 4.6 mm, LC column (Phenomenex, Torrance, CA, USA) with a Waters 2695 Separation Module (Milfold, MA, USA) with a Waters 996 Photodiode Array Detector. Separations were performed with a linear gradient from 0–60% ACN in the presence of 0.1% TFA over 20 min at a constant flow rate of 2 mL/min monitoring the peaks by UV detection (λ = 220 nm).

Subsequently, the obtained fractions were analysed with a MALDI-TOF/TOF 4800 mass spectrometer (AB Sciex, Framingham, MA, USA). After an initial full MS scan, enlargements on the MS signals of interest were acquired. For the basolateral fractions, the samples, after lyophilisation, were dissolved in 50 μL of 25% ACN and 0.1% TFA. The analysis was then performed on 2 μL of these solutions mixed with 2 μL of peptide MALDI matrix α-cyano-4-hydroxycynnamic acid (10 mg/mL aqueous 70% acetonitrile/0.1% TFA). The following analytical conditions were set for MALDI-TOF spectra acquisition: positive ion reflector mode, initial mass range 500–3500 Da (the mass range was then adapted for each enlargement), variable laser intensity (3000–3800), shots/sub-spectrum 50, total shots/spectrum 1500 and accelerating voltage of 20 kV. Final data analysis and fragments identification were done on Data Explorer software (AB Sciex, Framingham, MA, USA), utilizing external mass calibration performed with mass peptide standards (Sigma-Aldrich, St Louis, MO, USA).
