title: Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and 15N-metabolic labeling.
abstract: We describe the combined use of 15N-metabolic labeling and a cysteine-reactive biotin affinity tag to isolate and quantitate cysteine-containing polypeptides (Cys-polypeptides) from Deinococcus radiodurans as well as from mouse B16 melanoma cells. D. radiodurans were cultured in both natural isotopic abundance and 15N-enriched media. Equal numbers of cells from both cultures were combined and the soluble proteins extracted. This mixture of isotopically distinct proteins was derivatized using a commercially available cysteine-reactive reagent that contains a biotin group. Following trypsin digestion, the resulting modified peptides were isolated using immobilized avidin. The mixture was analyzed by capillary reversed-phase liquid chromatography (LC) online with ion trap mass spectrometry (MS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cyspolypeptides whose mass difference corresponds to the number of nitrogen atoms present in each of the peptides. Designation of Cys-polypeptide pairs is also facilitated by the distinctive isotopic distribution of the 15N-labeled peptides versus their 14N-labeled counterparts. Studies with mouse B16 cells maintained in culture allowed the observation of hundreds of isotopically distinct pairs of peptides by LC-FTICR analysis. The ratios of the areas of the pairs of isotopically distinct peptides showed the expected 1:1 labeling of the 14N and 15N versions of each peptide. An additional benefit from the present strategy is that the 15N-labeled peptides do not display significant isotope-dependent chromatographic shifts from their 14N-labeled counterparts, therefore improving the precision for quantitating peptide abundances. The methodology presented offers an alternate, cost-effective strategy for conducting global, quantitative proteomic measurements.
authors.affiliation: Environmental and Molecular Sciences Laboratory and Molecular Biosciences Department, Pacific Northwest National Laboratory, Richland, Washington 99352, USA.
authors.forename: T P
authors.initials: TP
authors.lastname: Conrads
authors.forename: K
authors.initials: K
authors.lastname: Alving
authors.forename: T D
authors.initials: TD
authors.lastname: Veenstra
authors.forename: M E
authors.initials: ME
authors.lastname: Belov
authors.forename: G A
authors.initials: GA
authors.lastname: Anderson
authors.forename: D J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: M S
authors.initials: MS
authors.lastname: Lipton
authors.forename: L
authors.initials: L
authors.lastname: Pasa-Tolić
authors.forename: H R
authors.initials: HR
authors.lastname: Udseth
authors.forename: W B
authors.initials: WB
authors.lastname: Chrisler
authors.forename: B D
authors.initials: BD
authors.lastname: Thrall
authors.forename: R D
authors.initials: RD
authors.lastname: Smith
mesh_terms: D000818:Animals; D001360:Avidin / Q000378:metabolism; D001419:Bacteria / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002478:Cells, Cultured / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003545:Cysteine / Q000032:analysis; D008546:Melanoma, Experimental / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D009587:Nitrogen Isotopes; D010449:Peptide Mapping; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification*; D020543:Proteome / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014357:Trypsin / Q000737:chemistry
chemical_list: D009587:Nitrogen Isotopes; D010455:Peptides; D020543:Proteome; D001360:Avidin; D001710:Biotin; D014357:Trypsin; D003545:Cysteine