Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites
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作者:
Ficarro, SB
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Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USANovartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
Ficarro, SB
[1
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Salomon, AR
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Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USANovartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
Salomon, AR
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Brill, LM
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Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USANovartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
Brill, LM
[1
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Mason, DE
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Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USANovartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
Mason, DE
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Stettler-Gill, M
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Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USANovartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
Stettler-Gill, M
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Brock, A
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Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USANovartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
Brock, A
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Peters, EC
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Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USANovartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
Peters, EC
[1
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机构:
[1] Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
A versatile integrated system has been developed for the automated enrichment and analysis of phosphopeptides by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This system utilizes two independently controlled high-performance liquid chromatography (HPLC) pumps, an autosampler and microvalves to prepare and elute samples into an ion trap mass spectrometer. The use of robust reversed-phase HPLC columns with integrated ESI emitter tips enables the reproducible detection and identification of low-femtomole quantities of phosphopeptides. The entire system is coordinated through a simple user interface by customized software. The ruggedness of the system is demonstrated by highly reproducible analyses of single and multi-protein digests, while its utility is demonstrated by the thorough evaluation of the relative immunoprecipitation efficiencies of several commercially available anti-phosphotyrosine antibodies. Copyright (C) 2004 John Wiley Sons, Ltd.