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Enhanced phosphopeptide isolation by Fe(III)‐IMAC using 1,1,1,3,3,3‐hexafluoroisopropanol
Author(s) -
Barnouin Karin N.,
Hart Sarah R.,
Thompson Andrew J.,
Okuyama Masahiro,
Waterfield Michael,
Cramer Rainer
Publication year - 2005
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200401323
Subject(s) - phosphopeptide , chemistry , steric effects , peptide , phosphate buffered saline , proteolysis , chromatography , phosphate , amino acid , combinatorial chemistry , biochemistry , stereochemistry , enzyme
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‐hexafluoroisopropanol is shown here to significantly improve Fe(III)‐IMAC enrichment and subsequent detection of phosphopeptides by MALDI‐MS.