title: Ion-trap mass spectrometry used in combination with gas chromatography for high-resolution metabolic flux determination.
abstract: Metabolic fluxes provide a detailed metric of the cellular metabolic state. Fluxes are estimated indirectly from available measurements, and various methods have been developed for this purpose. Of particular interest are methods that make use of stable isotopic tracers because they enable flux estimation at a fine resolution. In this report, we present a protocol for the use of ion-trap mass spectrometry (MS) in combination with gas chromatography to measure the mass isotopomer distribution of biomass hydrolysates. At physiological steady-state, these measurements directly reflect the isotopic tracer distribution in the amino acid central carbon metabolism precursors. Because the extent to which a metabolic flux network can be accurately resolved strongly depends on the reliability and precision of the MS measurements and, in light of the current need for quantitative high-throughput biological analysis at the microscale, we discuss every step of the measurement process, indicate possible sources of error, and suggest solutions to avoid them. Potential advantages to using ion-trap versus quadrupole MS are also addressed. The final protocol requires 0.5 mg of dry biomass to detect the mass isotopomer distribution of 2-4 fragments of 13 amino acids, with a relative variance less than 1% for the most abundant peaks.
authors.affiliation: Massachusetts Institute of Technology, Cambridge, MA, USA.
authors.forename: Maria I
authors.initials: MI
authors.lastname: Klapa
authors.forename: Juan-Carlos
authors.initials: JC
authors.lastname: Aon
authors.forename: Gregory
authors.initials: G
authors.lastname: Stephanopoulos
mesh_terms: D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D019149:Bioreactors; D002247:Carbon Isotopes; D003198:Computer Simulation; D003352:Corynebacterium / Q000737:chemistry / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007553:Isotope Labeling / Q000379:methods*; D008660:Metabolism / Q000502:physiology*; D008954:Models, Biological; D011786:Quality Control; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002247:Carbon Isotopes; D005947:Glucose