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Rapid Commun Mass Spectrom. 2005;19(1):57-71.

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

Author information

  • 1Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA. sficarro@gnf.org

Abstract

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.

PMID:
15570572
[PubMed - indexed for MEDLINE]
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