title: Analysis of isotopic labeling in peptide fragments by tandem mass spectrometry.
abstract: Phenotype in multicellular organisms is the consequence of dynamic metabolic events that occur in a spatially dependent fashion. This spatial and temporal complexity presents challenges for investigating metabolism; creating a need for improved methods that effectively probe biochemical events such as amino acid biosynthesis. Isotopic labeling can provide a temporal-spatial recording of metabolic events through, for example, the description of enriched amino acids in the protein pool. Proteins are therefore an important readout of metabolism and can be assessed with modern mass spectrometers. We compared the measurement of isotopic labeling in MS2 spectra obtained from tandem mass spectrometry under either higher energy collision dissociation (HCD) or collision induced dissociation (CID) at varied energy levels. Developing soybean embryos cultured with or without 13C-labeled substrates, and Escherichia coli MG1655 enriched by feeding 7% uniformly labeled glucose served as a source of biological material for protein evaluation. CID with low energies resulted in a disproportionate amount of heavier isotopologues remaining in the precursor isotopic distribution. HCD resulted in fewer quantifiable products; however deviation from predicted distributions were small relative to the CID-based comparisons. Fragment ions have the potential to provide information on the labeling of amino acids in peptides, but our results indicate that without further development the use of this readout in quantitative methods such as metabolic flux analysis is limited.
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Plant Genetic Research Unit, St. Louis, Missouri, United States of America; Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America.
authors.forename: Doug K
authors.initials: DK
authors.lastname: Allen
authors.affiliation: Donald Danforth Plant Science Center, St. Louis, Missouri, United States of America.
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Evans
authors.affiliation: Department of Plant Biology, University of Minnesota, St. Paul, Minnesota, United States of America.
authors.forename: Igor G L
authors.initials: IG
authors.lastname: Libourel
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000737:chemistry*; D007553:Isotope Labeling; D007554:Isotopes / Q000737:chemistry*; D013058:Mass Spectrometry; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D013025:Glycine max / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; D007554:Isotopes; D010446:Peptide Fragments; D010455:Peptides