*4.5. Preparation of Proteins from a Polyacrylamide Gel for Mass Spectrometric Analysis*

Proteins were fractionated on a SDS-polyacrylamide gel. After Coomassie blue R-250 staining, protein samples were extracted from the gel as described in Reference [68] with minor changes. Protein bands were excised from the gel with a razor, and the gel was then destained twice with 200 μL of 25 mM ammonium bicarbonate and 50% acetonitrile solution for 30 min at 37 ◦C. Protein disulfide bonds were reduced with 40 μL of 10 mM dithiothreitol (DTT) for 45 min 60 ◦C, followed by incubation with 30 μL of 100 mM iodoacetamide for 1 h at room temperature in the dark to alkylate free cysteines. Gel slices were washed again twice with 100 μL 25 mM ammonium bicarbonate and 50% acetonitrile solution for 15 min at 37 ◦C, dehydrated by adding 50 μL 100% acetonitrile and dried using a vacuum centrifuge. Gel pieces were incubated with up to 40 μL of activated trypsin (10 ng/μL) at 37 ◦C overnight. The next day, the supernatant was saved and the peptides were extracted from the gel by incubating gel slices in two consecutive changes of 50 μL of 5% trifluoroacetic acid and 50% acetonitrile solution for 1 h at 37 ◦C. Combined supernatants were dried using a vacuum centrifuge at 30 ◦C. Lyophilized peptides were dissolved in 20 μL of 0.1% trifluoroacetic acid solution. Peptides purified and concentrated using Millipore C18 ZipTips.

#### *4.6. Enzyme Assay*

Activity of 3-(2,4-dihydroxyphenyl)-propionic acid hydroxylase was measured spectrophotometrically by monitoring absorption changes of the reaction mixture at 340 nm wavelength due to the oxidation of either NADH or NADPH (ε340 = 6220 M−<sup>1</sup> cm−1) after the addition of the substrate. The activity measurements were made with cell-free extracts or the purified protein. All measurements of the enzyme activity were carried out at room temperature in 1 mL of reaction mixture, containing 25–50 mM tricine or potassium phosphate buffers (pH 7.8), 100 μM NADH or NADPH, and 150 μM aromatic substrate. One unit of activity was defined as the amount of the enzyme that catalyzed the oxidation of 1 μmol of NADH or NADPH per minute.
