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

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