Automated immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry platform for profiling protein phosphorylation sites.
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.
Ficarro
Scott B
SB
Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, CA 92121, USA. sficarro@gnf.org
Salomon
Arthur R
AR
Brill
Laurence M
LM
Mason
Daniel E
DE
Stettler-Gill
Michelle
M
Brock
Ansgar
A
Peters
Eric C
EC
D000595:Amino Acid Sequence; D001331:Automation; D001665:Binding Sites; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry*; D010766:Phosphorylation; D040081:Protein Array Analysis; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009842:Oligopeptides; D010750:Phosphoproteins; D012710:Serum Albumin, Bovine