title: Quantification of peptide m/z distributions from 13C-labeled cultures with high-resolution mass spectrometry.
abstract: Isotopic labeling studies of primary metabolism frequently utilize GC/MS to quantify (13)C in protein-hydrolyzed amino acids. During processing some amino acids are degraded, which reduces the size of the measurement set. The advent of high-resolution mass spectrometers provides a tool to assess molecular masses of peptides with great precision and accuracy and computationally infer information about labeling in amino acids. Amino acids that are isotopically labeled during metabolism result in labeled peptides that contain spatial and temporal information that is associated with the biosynthetic origin of the protein. The quantification of isotopic labeling in peptides can therefore provide an assessment of amino acid metabolism that is specific to subcellular, cellular, or temporal conditions. A high-resolution orbital trap was used to quantify isotope labeling in peptides that were obtained from unlabeled and isotopically labeled soybean embryos and Escherichia coli cultures. Standard deviations were determined by estimating the multinomial variance associated with each element of the m/z distribution. Using the estimated variance, quantification of the m/z distribution across multiple scans was achieved by a nonlinear fitting approach. Observed m/z distributions of uniformly labeled E. coli peptides indicated no significant differences between observed and simulated m/z distributions. Alternatively, amino acid m/z distributions obtained from GC/MS were convolved to simulate peptide m/z distributions but resulted in distinct profiles due to the production of protein prior to isotopic labeling. The results indicate that peptide mass isotopologue measurements faithfully represent mass distributions, are suitable for quantification of isotope-labeling-based studies, and provide additional information over existing methods.
authors.affiliation: Plant Genetic Research Unit, Agricultural Research Service, U.S. Department of Agriculture (USDA-ARS), Donald Danforth Plant Science Center , 975 North Warson Road, St. Louis, Missouri 63132, United States.
authors.forename: Doug K
authors.initials: DK
authors.lastname: Allen
authors.forename: Joshua
authors.initials: J
authors.lastname: Goldford
authors.forename: James K
authors.initials: JK
authors.lastname: Gierse
authors.forename: Dominic
authors.initials: D
authors.lastname: Mandy
authors.forename: Christine
authors.initials: C
authors.lastname: Diepenbrock
authors.forename: Igor G L
authors.initials: IG
authors.lastname: Libourel
mesh_terms: D000595:Amino Acid Sequence; D018533:Biomass; D002247:Carbon Isotopes; D046508:Culture Techniques; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism; D012639:Seeds / Q000254:growth & development / Q000378:metabolism; D013025:Glycine max / Q000254:growth & development / Q000378:metabolism
chemical_list: D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D010940:Plant Proteins