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

Proteomics

metabolism, Trypsin, Cyclic AMP-Dependent Protein Kinases, Humans, Metals, Chromatography, Affinity, Phosphopeptides, Phosphoproteins, chemistry, genetics, Phosphorylation, Recombinant Proteins, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Telomeric Repeat Binding Protein 1, Adenosine Triphosphate

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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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      Journal
      10.1002/pmic.200500223
      16342239

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