Analysis of amino acid isotopomers using FT-ICR MS.
Fluxes through known metabolic pathways and the presence of novel metabolic reactions are often determined by feeding isotopically labeled substrate to an organism and then determining the isotopomer distribution in amino acids in proteins. However, commonly used techniques to measure the isotopomer distributions require derivatization prior to analysis (gas chromatography/mass spectrometry (GC/MS)) or large sample sizes (nuclear magnetic resonance (NMR) spectroscopy). Here, we demonstrate the use of Fourier transform-ion cyclotron resonance mass spectrometry with direct infusion via electrospray ionization to rapidly measure the amino acid isotopomer distribution in a biomass hydrolysate of the soil bacterium Desulfovibrio vulgaris Hildenborough. By applying high front-end resolution for the precursor ion selection followed by sustained off-resonance irradiation collision-induced dissociation, it was possible to determine exactly and unambiguously the specific locations of the labeled atoms in the amino acids, which usually requires a combination of 2-D 13C NMR spectroscopy and GC/MS. This method should be generally applicable to all biomass samples and will allow more accurate determination of metabolic fluxes with less work and less sample.
Pingitore
Francesco
F
Virtual Institute of Microbial Stress and Survival, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
Tang
Yinjie
Y
Kruppa
Gary H
GH
Keasling
Jay D
JD
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry*; D002244:Carbon / Q000737:chemistry; D002247:Carbon Isotopes / Q000737:chemistry; D016969:Desulfovibrio vulgaris / Q000737:chemistry / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D053858:Metabolic Networks and Pathways; D015394:Molecular Structure; D062907:Oxaloacetic Acid / Q000737:chemistry / Q000378:metabolism; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D000596:Amino Acids; D002247:Carbon Isotopes; D011522:Protons; D062907:Oxaloacetic Acid; D002244:Carbon