Crocetin dialdehyde was dissolved in 100 μL of incubation buffer (200 mm pyrophosphate, 200 mm NaCl, pH 7.5, 2 μL of 100 mm NAD+), and 80 μL of crude protein and water were added to give a total volume of 200 μL. Incubations were performed at 28 °C for 30 min, stopped by addition of 1 mL of acetone, extracted with light petroleum/diethyl ether (1:4, v/v). The reactions were analyzed by LC-APCI(+)-MS, using a Q-exactive quadrupole Orbitrap mass spectrometry system (ThermoFisher Scientific, Madrid, Spain), coupled to a HPLC system equipped with a photodiode array detector (Dionex, Madrid, Spain) as described [9 (link)].
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