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Phosphopeptide detection using automated online IMAC-capillary LC-ESI-MS/MS.

Jinglan Wang·Yangjun Zhang·Hong Jiang·Yun Cai·Xiaohong Qian
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Abstract

IMAC has become a commonly used technique in phosphoprotein analysis because of its affinity for phosphopeptides. However, the commonly used strategy combining offline IMAC enrichment with desalting procedures prior to MS/MS makes this method laborious. Here we report the development of a robust and automatic IMAC-capillary RP HPLC-ESI MS/MS technology platform, by which all procedures needed in phosphopeptide analysis including IMAC enrichment, RP HPLC separation and nanospray MS/MS can be done automatically controlled by the MassLynx program. The platform was optimized by analyzing standard phosphopeptide, and was then applied to the identification of phosphorylation sites of recombinant human telomeric repeat binding factor 1 treated with kinase in vitro, and two phosphorylation sites are defined.

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Adenosine TriphosphateChromatography, AffinityCyclic AMP-Dependent Protein KinasesHumansMetalsPhosphopeptidesPhosphoproteinsPhosphorylationRecombinant ProteinsSpectrometry, Mass, Electrospray IonizationSpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationTelomeric Repeat Binding Protein 1TrypsinJournal ArticleResearch Support, Non-U.S. Gov't

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Jinglan Wang, Yangjun Zhang, Hong Jiang, Yun Cai, Xiaohong Qian. 2006. Phosphopeptide detection using automated online IMAC-capillary LC-ESI-MS/MS.. https://doi.org/10.1002/pmic.200500223

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