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XB-ART-40932
J Biochem 2010 May 01;1475:689-96. doi: 10.1093/jb/mvp211.
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A fully automated phosphopeptide purification system for large-scale phosphoproteome analysis.

Iwase Y , Honma S , Matsuzaki M , Miyakawa Y , Kanno T , Ishii K , Furuichi N , Furukawa K , Horigome T .


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For large-scale phosphoproteome analysis based on mass spectrometry, a fully automated phosphopeptide purification system is essential to obtain reproducible results. An automated system involving pre-cleaning of a sample with a polymer-based reversed-phase column, phosphopeptide purification with a titania column and analysis of the phosphopeptide fraction with a reversed-phase column was developed, and then the analytical conditions for a complex peptide mixture were optimized. A lower flow rate for application of samples to the titania column was essential to obtain high recoveries of phosphopeptides from complex protein digests. Washing with 1 M NaCl and 2-propanol, and two cycles of washing with four solvents for the titania column were necessary to minimize non-phosphorylated peptides in the phosphopeptide fraction. Using this system under the optimized conditions, a peptide fraction including >90% phosphopeptides could be obtained highly reproducibly from a tryptic digest of a complex protein mixture, i.e. a Xenopus egg cytosol fraction, without any pre-treatment.

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???displayArticle.link??? J Biochem