Mass-Spectrometry-Based Quantification of Protein-Bound Fatty Acid Synthesis Intermediates from Escherichia coli.
The production of fatty acids from simple nutrients occurs via a complex biosynthetic pathway with dozens of intermediate compounds and multiple branch points. Despite its importance for microbial physiology and biotechnology, critical aspects of fatty acid biosynthesis, especially dynamics of in vivo regulation, remain poorly characterized. We have developed a liquid chromatography/mass spectroscopy (LC-MS) method for relative quantification of fatty acid synthesis intermediates in Escherichia coli, a model organism for studies of fatty acid metabolism. The acyl carrier protein, a vehicle for the substrates and intermediates of fatty acid synthesis, is extracted from E. coli, proteolytically digested, resolved using reverse-phase LC, and detected using electrospray ionization coupled with a tandem MS. Our method reliably resolves 21 intermediates of fatty acid synthesis, with an average relative standard deviation in ratios of individual acyl-ACP species to total ACP concentrations of 20%. We demonstrate that fast sampling and quenching of cells is essential to accurately characterize intracellular concentrations of ACP species. We apply our method to examine the rapid response of fatty acid metabolism to the antibiotic cerulenin. We anticipate that our method will enable the characterization of in vivo regulation and kinetics of microbial fatty acid synthesis at unprecedented detail and will improve integration of fatty acid synthesis into models of microbial metabolism.
Department of Bionanoscience, Delft University of Technology, Kavli Institute of Nanoscience Delft , Lorentzweg 1, 2628CJ Delft, The Netherlands.
Marek J
MJ
Noga
Department of Bionanoscience, Delft University of Technology, Kavli Institute of Nanoscience Delft , Lorentzweg 1, 2628CJ Delft, The Netherlands.
Mattia
M
Cerri
Department of Bionanoscience, Delft University of Technology, Kavli Institute of Nanoscience Delft , Lorentzweg 1, 2628CJ Delft, The Netherlands.
Nicole
N
Imholz
Department of Bionanoscience, Delft University of Technology, Kavli Institute of Nanoscience Delft , Lorentzweg 1, 2628CJ Delft, The Netherlands.
Pawel
P
Tulinski
Department of Bionanoscience, Delft University of Technology, Kavli Institute of Nanoscience Delft , Lorentzweg 1, 2628CJ Delft, The Netherlands.
Enes
E
Şahin
Department of Bionanoscience, Delft University of Technology, Kavli Institute of Nanoscience Delft , Lorentzweg 1, 2628CJ Delft, The Netherlands.
Gregory
G
Bokinsky
D000213:Acyl Carrier Protein / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism; D002569:Cerulenin / Q000494:pharmacology; D004926:Escherichia coli / Q000737:chemistry*; D005227:Fatty Acids / Q000096:biosynthesis / Q000378:metabolism*; D013058:Mass Spectrometry; D011485:Protein Binding
D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D002352:Carrier Proteins; D005227:Fatty Acids; D002569:Cerulenin
acyl carrier protein; biofuels; fatty acids; mass spectrometry; metabolomics; phospholipids; phosphopantetheine; post-translational modification; quantitative proteomics; thioesters