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Titolo:
Two-dimensional electrophoretic/chromatographic separations combined with electrospray ionization FTICR mass spectrometry for high throughput proteome analysis
Autore:
Gao, HY; Shen, YF; Veenstra, TD; Harkewicz, R; Anderson, GA; Bruce, JE; Pasa-Tolic, L; Smith, RD;
Indirizzi:
Battelle Mem Inst, Pacific NW Natl Lab, Environm Mol Sci lab, Richland, WA99352 USA Battelle Mem Inst Richland WA USA 99352 ol Sci lab, Richland, WA99352 USA
Titolo Testata:
JOURNAL OF MICROCOLUMN SEPARATIONS
fascicolo: 7, volume: 12, anno: 2000,
pagine: 383 - 390
SICI:
1040-7685(2000)12:7<383:TESCWE>2.0.ZU;2-A
Fonte:
ISI
Lingua:
ENG
Soggetto:
SEQUENCE DATABASES; PROTEINS; IDENTIFICATION; ELECTROPHORESIS; MIXTURES; BIOLOGY; GENOME;
Keywords:
proteomics; two-dimensional separations; CIEF; HPLC-FTICR;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Physical, Chemical & Earth Sciences
Citazioni:
27
Recensione:
Indirizzi per estratti:
Indirizzo: Smith, RD Battelle Mem Inst, Pacific NW Natl Lab, Environm Mol Sci lab, POB 999,MSINK8-98, Richland, WA 99352 USA Battelle Mem Inst POB 999,MSIN K8-98 Richland WA USA 99352 2 USA
Citazione:
H.Y. Gao et al., "Two-dimensional electrophoretic/chromatographic separations combined with electrospray ionization FTICR mass spectrometry for high throughput proteome analysis", J MICROCOL, 12(7), 2000, pp. 383-390

Abstract

A two-dimensional separation strategy combined with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR) is being developed for high throughput proteomic analyses. Capillary isoelectric focusing (CIEF) coupled online with a robotic fraction collector is used to separate and collect microliter fractions of soluble Saccharomyces cerevisiae (yeast) proteins eluting from the capillary into a microtiter plate. Following tryptic digestion of each fraction, the resultant peptides areseparated using capillary high performance liquid chromatography (HPLC) and analyzed by online ESI-FTICR. Protein identification is based upon the use of the experimentally measured peptide masses as accurate mass tags, augmented by conventional MS/MS methods as necessary, to identify proteins predicted from the yeast genome sequence. This new separation strategy is beingevaluated using proteins extracted from yeast grown in natural isotopic abundance and N-15-enriched media. Two isotopically distinct versions of eachpeptide are thus observed in the ESI-FTICR spectra. The mass differences between the two versions are used to determine the number of nitrogen atoms in the peptide, and provide an additional constraint that aids protein identification. More importantly, the use of this stable-isotope labeling strategy enables the generation of "comparative displays" of the precise relative protein abundances. This two-dimensional separation strategy combined with ESI-FTICR analysis is expected to be highly amenable to automation. (C) 2000 John Wiley & Sons, Inc.

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Documento generato il 12/07/20 alle ore 06:07:58