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Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol

Barnouin, K.N., Hart, S.R., Thompson, A.J., Okuyama, M., Waterfield, M. and Cramer, R. (2005) Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol. Proteomics, 5 (17). pp. 4376-4388. ISSN 1615-9853

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To link to this article DOI: 10.1002/pmic.200401323

Abstract/Summary

IMAC can be used to selectively enrich phosphopeptides from complex peptide mixtures, but co-retention of acidic peptides together with the failure to retain some phosphopeptides restricts the general utility of the method. In this study Fe(III)-IMAC was qualitatively and quantitatively assessed using a panel of phosphopeptides, both synthetic and derived from proteolysis of known phosphoproteins, to identify the causes of success and failure in the application of this technique. Here we demonstrate that, as expected, peptides with a more acidic amino acid content are generally more efficiently purified and detected by MALDI-MS after Fe(III)-IMAC than those with a more basic content. Modulating the loading buffer used for Fe(III)-IMAC significantly affects phosphopeptide binding and suggests that conformational factors that lead to steric hindrance and reduced accessibility to the phosphate are important. The use of 1,1,1,3,3,3-hexa-fluoroisopropanol is shown here to significantly improve Fe(III)-IMAC enrichment and subsequent detection of phosphopeptides by MALDI-MS.

Item Type:Article
Refereed:Yes
Divisions:Faculty of Life Sciences > School of Chemistry, Food and Pharmacy > Department of Chemistry
ID Code:10992
Uncontrolled Keywords:1,1,1,3,3,3-hexafluoroisopropanol, IMAC, MS, phosphopeptide, phosphorylation , ION AFFINITY-CHROMATOGRAPHY, PROTEIN-KINASE CK2, TANDEM MASS-SPECTROMETRY, IMMOBILIZED IRON(III) IONS, PHOSPHORYLATION SITES, RETINOBLASTOMA PROTEIN, SUBSTRATE-SPECIFICITY, ACTIN POLYMERIZATION, HISTONE H1, CYCLIN-E

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