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Titolo:
Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and N-15-Metabolic labeling
Autore:
Conrads, TP; Alving, K; Veenstra, TD; Belov, ME; Anderson, GA; Anderson, DJ; Lipton, MS; Pasa-Tolic, L; Udseth, HR; Chrisler, WB; Thrall, BD; Smith, RD;
Indirizzi:
Pacific NW Natl Lab, Mol Biosci Dept, Richland, WA 99352 USA Pacific NW Natl Lab Richland WA USA 99352 ci Dept, Richland, WA 99352 USA Pacific NW Natl Lab, Environm & Mol Sci Lab, Richland, WA 99352 USA Pacific NW Natl Lab Richland WA USA 99352 Sci Lab, Richland, WA 99352 USA
Titolo Testata:
ANALYTICAL CHEMISTRY
fascicolo: 9, volume: 73, anno: 2001,
pagine: 2132 - 2139
SICI:
0003-2700(20010501)73:9<2132:QAOBAM>2.0.ZU;2-X
Fonte:
ISI
Lingua:
ENG
Soggetto:
RESONANCE MASS-SPECTROMETRY; LINEAR ION-TRAP; ELECTROSPRAY-IONIZATION; PROTEIN EXPRESSION; ELECTROPHORESIS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Physical, Chemical & Earth Sciences
Citazioni:
27
Recensione:
Indirizzi per estratti:
Indirizzo: Smith, RD Pacific NW Natl Lab, Mol Biosci Dept, POB 999,MSIN K8-98, Richland, WA 99352 USA Pacific NW Natl Lab POB 999,MSIN K8-98 Richland WA USA 99352 USA
Citazione:
T.P. Conrads et al., "Quantitative analysis of bacterial and mammalian proteomes using a combination of cysteine affinity tags and N-15-Metabolic labeling", ANALYT CHEM, 73(9), 2001, pp. 2132-2139

Abstract

We describe the combined use of N-15-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 N-15-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) on-line with ion trap mass spectrometry (RIS) as well as Fourier transform ion cyclotron resonance (FTICR) MS. The resulting spectra contain numerous pairs of Cys-polypeptides 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 N-15-labeled peptides versus their N-14-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:I labeling of the N-14 arid N-15 versions of each peptide. An additional benefit from the present strategy Is that the N-15-labeled peptides do not display significant isotope-dependent chromatographicshifts from their N-14-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.

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Documento generato il 24/09/20 alle ore 05:09:56