Figure 2

Scheme of the transport quantification method by MALDI-TOF MS. The in vitro BBB cell-based model (a) is performed in a transwell system where a membrane cultured with endothelial cells delimits two compartments (donor and acceptor). The donor compartment contains the light peptide before starting the experiment; at the end of the assay, a certain amount of peptide has been transported to the acceptor compartment (if 200 μM is assayed in the donor compartment, around 2 μM needs to be quantified in the acceptor compartment). Thus, (b) two aliquots of the heavy peptide are prepared, one at 200 μM and another at 2 μM; 10 μL of the assayed light peptide (from acceptor or donor compartments) and 10 μL of the heavy version, at similar concentrations, are mixed. Subsequently, 1 μL of this mixture and 1 μL of an appropriate MALDI matrix (e.g. ACH matrix solution) are placed on a MALDI plate. Finally, the spectra are acquired. A spectrum (c) obtained from a solution containing light and heavy versions of 9D peptide at 2 μM is shown. Light and heavy peptides are observed as the m/z of the peptides plus H⁺, Na⁺ or K⁺. In all cases, isotopic homolog peaks between light and heavy peptides display a 4-amu mass difference.