title: Analysis of carbon and nitrogen co-metabolism in yeast by ultrahigh-resolution mass spectrometry applying 13C- and 15N-labeled substrates simultaneously.
abstract: Alternative metabolic pathways inside a cell can be deduced using stable isotopically labeled substrates. One prerequisite is accurate measurement of the labeling pattern of targeted metabolites. Experiments are generally limited to the use of single-element isotopes, mainly (13)C. Here, we demonstrate the application of direct infusion nanospray, ultrahigh-resolution Fourier transform ion cyclotron resonance-mass spectrometry (FTICR-MS) for metabolic studies using differently labeled elemental isotopes simultaneously--i.e., (13)C and (15)N--in amino acids of a total protein hydrolysate. The optimized strategy for the analysis of metabolism by a hybrid linear ion trap-FTICR-MS comprises the collection of multiple adjacent selected ion monitoring scans. By limiting both the width of the mass range and the number of ions entering the ICR cell with automated gain control, sensitive measurements of isotopologue distribution were possible without compromising mass accuracy and isotope intensity mapping. The required mass-resolving power of more than 60,000 is only achievable on a routine basis by FTICR and Orbitrap mass spectrometers. Evaluation of the method was carried out by comparison of the experimental data to the natural isotope abundances of selected amino acids and by comparison to GC/MS results obtained from a labeling experiment with (13)C-labeled glucose. The developed method was used to shed light on the complexity of the yeast Saccharomyces cerevisiae carbon-nitrogen co-metabolism by administering both (13)C-labeled glucose and (15)N-labeled alanine. The results indicate that not only glutamate but also alanine acts as an amino donor during alanine and valine synthesis. Metabolic studies using FTICR-MS can exploit new possibilities by the use of multiple-labeled elemental isotopes.
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical and Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Lars M
authors.initials: LM
authors.lastname: Blank
authors.forename: Rahul R
authors.initials: RR
authors.lastname: Desphande
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
authors.forename: Heiko
authors.initials: H
authors.lastname: Hayen
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism*; D002247:Carbon Isotopes / Q000032:analysis / Q000378:metabolism; D017357:Cyclotrons; D004867:Equipment Design; D005583:Fourier Analysis; D007553:Isotope Labeling / Q000379:methods; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods*; D053858:Metabolic Networks and Pathways; D009584:Nitrogen / Q000378:metabolism*; D009587:Nitrogen Isotopes / Q000032:analysis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D002244:Carbon; D009584:Nitrogen