GNPS Human AKR1C3 Binds Agonists of GPR84 and Participates in an Expanded Polyamine Pathway (tandem MS files of murine type I fatty acid synthase NADPH assay for alpha-pyrone biosynthesis)

Representative tandem MS files generated from mouse type I fatty acid synthase biochemical assay with NADPH titration.

Protocol description: Mouse FAS was enriched using dual-affinity chromatography (Ni-NTA, nitrilotriacetic acid, Strep-Tactin XT Sepharose from Cytiva) and subjected to in vitro enzymatic assays. In this assay, we titrated different amounts of NADPH (0-350 uM) in the reaction mixture containing malonyl-CoA (175 uM), acetyl-CoA (25 uM), and enriched mFAS (6.2 mg/mL) in PBS buffer (pH 7.4) After 50 minutes of incubation, the reactions were quenched with acetonitrile, and the mixtures were centrifuged and subjected to HPLC-HR-QTOF-MS analysis (positive mode). We observed that with no NADPH we got the highest production of TAL (triacetic acid lactone), with high NADPH (350 uM) we got the highest production of palmitate, and that middle ranges of NADPH would variably generate the alpha-pyrone family.
File description:
in-vitro-NADPH-0-1msms: a representative tandem MS file of alpha pyrones from mFAS in vitro biochemical assay (as described above) titrated with 0 uM NADPH co-factor.
in-vitro-NADPH-100-1msms: a representative tandem MS file of alpha pyrones from mFAS in vitro biochemical assay (as described above) titrated with 100 uM NADPH co-factor.
in-vitro-NADPH-200-1msms: a representative tandem MS file of alpha pyrones from mFAS in vitro biochemical assay (as described above) titrated with 200 uM NADPH co-factor.
Reversed-phase chromatography was performed with a Kinetex (Cat. # 00G-4601-E0) 5 um C18 100 A column (250 by 4.6 mm), using a water:acetonitrile gradient containing 0.1% formic acid at 0.7 mL/min flow rate: 0-30 min, 10% to 100% acetonitrile. Positive mode (qTOF)