Enhanced phosphopeptide isolation by Fe(III)-IMAC using 1,1,1,3,3,3-hexafluoroisopropanol.
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.
Barnouin
Karin N
KN
The Ludwig Institute for Cancer Research, Proteomics Unit, UCL branch, London, UK. karin.barnouin@cancer.org.uk
Hart
Sarah R
SR
Thompson
Andrew J
AJ
Okuyama
Masahiro
M
Waterfield
Michael
M
Cramer
Rainer
R
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
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