Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites

被引:77
作者
Ficarro, SB [1 ]
Salomon, AR [1 ]
Brill, LM [1 ]
Mason, DE [1 ]
Stettler-Gill, M [1 ]
Brock, A [1 ]
Peters, EC [1 ]
机构
[1] Novartis Res Fdn, Genom Inst, San Diego, CA 92121 USA
关键词
D O I
10.1002/rcm.1746
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:57 / 71
页数:15
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