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Open AccessArticle

A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry

1
Department of Biochemistry and Molecular Biology, Villum Center for Bioanalytical Sciences, University of Southern Denmark, 5000 Odense, Denmark
2
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany
*
Author to whom correspondence should be addressed.
Biomolecules 2020, 10(1), 118; https://doi.org/10.3390/biom10010118
Received: 20 December 2019 / Revised: 5 January 2020 / Accepted: 7 January 2020 / Published: 10 January 2020
(This article belongs to the Section Enzymology)
De novo fatty acid synthesis is a pivotal enzymatic process in all eukaryotic organisms. It is involved in the conversion of glucose and other nutrients to fatty acyl (FA) chains, that cells use as building blocks for membranes, energy storage, and signaling molecules. Central to this multistep enzymatic process is the cytosolic type I fatty acid synthase complex (FASN) which in mammals produces, according to biochemical textbooks, primarily non-esterified palmitic acid (NEFA 16:0). The activity of FASN is commonly measured using a spectrophotometry-based assay that monitors the consumption of the reactant NADPH. This assay is indirect, can be biased by interfering processes that use NADPH, and cannot report the NEFA chain-length produced by FASN. To circumvent these analytical caveats, we developed a simple mass spectrometry-based assay that affords monitoring of FASN activity and its product-specificity. In this assay (i) purified FASN is incubated with 13C-labeled malonyl-CoA, acetyl-CoA, and NADPH, (ii) at defined time points the reaction mixture is spiked with an internal NEFA standard and extracted, and (iii) the extract is analyzed directly, without vacuum evaporation and chemical derivatization, by direct-infusion high-resolution mass spectrometry in negative ion mode. This assay supports essentially noise-free detection and absolute quantification of de novo synthetized 13C-labled NEFAs. We demonstrate the efficacy of our assay by determining the specific activity of purified cow FASN and show that in addition to the canonical NEFA 16:0 this enzyme also produces NEFA 12:0, 14:0, 18:0, and 20:0. We note that our assay is generic and can be carried out using commonly available high-resolution mass spectrometers with a resolving power as low as 95,000. We deem that our simple assay could be used as high-throughput screening technology for developing potent FASN inhibitors and for enzyme engineering aimed at modulating the activity and the product-landscape of fatty acid synthases.
Keywords: fatty acid synthase; enzyme activity; high-resolution shotgun lipidomics; Orbitrap mass spectrometry fatty acid synthase; enzyme activity; high-resolution shotgun lipidomics; Orbitrap mass spectrometry
MDPI and ACS Style

Topolska, M.; Martínez-Montañés, F.; Ejsing, C.S. A Simple and Direct Assay for Monitoring Fatty Acid Synthase Activity and Product-Specificity by High-Resolution Mass Spectrometry. Biomolecules 2020, 10, 118.

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