Characterization of AANATL7-catalyzed Histamine Acetylation

A Phenomenex Kinetex 2.6 μm C₁₈ 100 Å (50 mm × 2.1 mm) reverse phase column coupled to an Agilent 6540 instrument using a liquid chromatography/quadrupole time-of-flight mass spectrometer (LC/QTOF-MS) in positive ion mode was used to characterize the AANATL7-catalyzed product from the substrates acetyl-CoA and histamine. An enzyme reaction mixture containing 300 mM Tris-HCl (pH 8.0), 500 μM acetyl-CoA, 50 mM histamine, and 9.3 μg of AANATL7 in a final volume of 750 μL was incubated for 30 min at 22 °C. Following the 30 min incubation period, AANATL7 was removed from the reaction mixture using a Millipore 10 kDa ultrafilter in which the reaction product flowed through the filter while AANATL7 was retained in the hold-up volume on the filter. The final reaction product was then injected onto the LC/QTOF-MS instrument to analyze the retention time and high-resolution mass. The resulting retention time and high-resolution mass for the reaction product were compared to those of a commercial standard of N^ω-acetylhistamine. The experimental conditions for LC/QTOF-MS analysis are reported in ref 4 (link).