title: Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol.
abstract: 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.
authors.affiliation: The Ludwig Institute for Cancer Research, Proteomics Unit, UCL branch, London, UK. karin.barnouin@cancer.org.uk
authors.forename: Karin N
authors.initials: KN
authors.lastname: Barnouin
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Hart
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Thompson
authors.forename: Masahiro
authors.initials: M
authors.lastname: Okuyama
authors.forename: Michael
authors.initials: M
authors.lastname: Waterfield
authors.forename: Rainer
authors.initials: R
authors.lastname: Cramer
mesh_terms: D000595:Amino Acid Sequence; D002364:Caseins / Q000737:chemistry; D018076:DNA, Complementary; D005982:Glutathione Transferase / Q000737:chemistry; D006801:Humans; D007093:Imidazoles; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation; D020005:Propanols; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D006845:Hydrocarbons, Fluorinated
chemical_list: D002364:Caseins; D018076:DNA, Complementary; D005982:Glutathione Transferase; D007093:Imidazoles; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010748:Phosphopeptides; D010750:Phosphoproteins; D020005:Propanols; D011994:Recombinant Proteins; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C005954:imidazoleacetic acid