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Titolo:
A simplified device for protein identification by microcapillary gradient liquid chromatography-tandem mass spectrometry
Autore:
McGinley, MD; Davis, MT; Robinson, JH; Spahr, CS; Bures, EJ; Beierle, J; Mort, J; Patterson, SD;
Indirizzi:
Amgen Inc, Biochem, Thousand Oaks, CA 91320 USA Amgen Inc Thousand Oaks CA USA 91320 Biochem, Thousand Oaks, CA 91320 USA
Titolo Testata:
ELECTROPHORESIS
fascicolo: 9, volume: 21, anno: 2000,
pagine: 1678 - 1684
SICI:
0173-0835(200005)21:9<1678:ASDFPI>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
SOLVENT DELIVERY SYSTEM; GEL-SEPARATED PROTEINS; ELECTROSPRAY INTERFACE; POLYACRYLAMIDE GELS; PEPTIDES; DIGESTION; MIXTURES; DATABASE; SEQUENCE;
Keywords:
mass spectrometry; protein identification; microelectrospray; capillary high performance liquid chromatography; proteomics;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
29
Recensione:
Indirizzi per estratti:
Indirizzo: Patterson, SD Amgen Inc, Biochem, 1 Amgen Ctr Dr MS 14-2-E, Thousand Oaks,CA 91320 USA Amgen Inc 1 Amgen Ctr Dr MS 14-2-E Thousand Oaks CA USA 91320
Citazione:
M.D. McGinley et al., "A simplified device for protein identification by microcapillary gradient liquid chromatography-tandem mass spectrometry", ELECTROPHOR, 21(9), 2000, pp. 1678-1684

Abstract

A simplified device and procedure have been developed for microcapillary gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS). This procedure has proved useful in identifying low level quantities of proteins from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gelbands. Microelectrospray needles are packed with reversed-phase resin and function both as a high performance liquid chromatography (HPLC) column anda nanospray mass spectrometer tip when interfaced between an HPLC and ion trap mass spectrometer. Variable submicroliter flow rates are generated by flow splitting between the microetectrospray capillary and an HPLG system. A manual injector is used to inject a protein digest mixture that binds to the column and is then washed at a high flow rate (2 mu L/min post split). Gradient elution of bound peptides was initiated by the injection of a filled loop of 70% v/v methanol (5 mu L) concomitant with a reduction of flow rate (0.1 mu/min post split). This forms a diffusion-dependent gradient of variable length (typically 15-30 min in length) depending upon the final flow rate. Chromatographic separations of a standard solution digest demonstrate that this diffusion-dependent gradient provides reasonable separations such that multiple peptide identifications by MS/MS can be obtained. Application of this methodology to the analysis of several in-gel-digested gel-separated proteins is presented to demonstrate its utility.

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Documento generato il 26/09/20 alle ore 05:27:24