title: Proteome analysis using selective incorporation of isotopically labeled amino acids.
abstract: A method is described for identifying intact proteins from genomic databases using a combination of accurate molecular mass measurements and partial amino acid content. An initial demonstration was conducted for proteins isolated from Escherichia coli (E. coli) using a multiple auxotrophic strain of K12. Proteins extracted from the organism grown in natural isotopic abundance minimal medium and also minimal medium containing isotopically labeled leucine (Leu-D10), were mixed and analyzed by capillary isoelectric focusing (CIEF) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FTICR). The incorporation of the isotopically labeled Leu residue has no effect on the CIEF separation of the protein, therefore both versions of the protein are observed within the same FTICR spectrum. The difference in the molecular mass of the natural isotopic abundance and Leu-D10 isotopically labeled proteins is used to determine the number of Leu residues present in that particular protein. Knowledge of the molecular mass and number of Leu residues present can be used to unambiguously identify the intact protein. Preliminary results show the efficacy of this method for unambiguously identifying proteins isolated from E. coli.
authors.affiliation: Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratories, Richland, Washington 99352, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: S
authors.initials: S
authors.lastname: Martinović
authors.forename: G A
authors.initials: GA
authors.lastname: Anderson
authors.forename: L
authors.initials: L
authors.lastname: Pasa-Tolić
authors.forename: R D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D000596:Amino Acids / Q000032:analysis*; D019075:Electrophoresis, Capillary; D004926:Escherichia coli / Q000737:chemistry*; D007525:Isoelectric Focusing; D007553:Isotope Labeling; D020543:Proteome / Q000032:analysis*; D011868:Radioisotopes / Q000032:analysis
chemical_list: D000596:Amino Acids; D020543:Proteome; D011868:Radioisotopes