Selective extraction and characterization of a histidine-phosphorylated peptide using immobilized copper(II) ion affinity chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Phosphorylation is the predominant posttranslational modification involved in regulating enzymatic activity and mediating signal transduction in prokaryotic and eukaryotic cells. Selective enrichment of phosphorylated peptides prior to mass spectrometric analysis facilitates identification of phosphorylated proteins, determination of specific phosphorylated residues, and characterization of the conditions under which phosphorylation occurs. Such protocols have been established for peptides containing residues that form phosphoesters, such as serine and threonine, using immobilized metal-ion affinity chromatography. Despite the importance of histidine phosphorylation in two-component signal transduction pathways, similar protocols for peptides containing phosphorylated histidine (P-His) residues have proven elusive, due to the instability of these modifications and the propensity of unphosphorylated histidines to interact with immobilized metals ions. We describe a method for the selective extraction of a P-His-containing peptide using immobilized copper(II) ions and disposable metal-chelating pipet tips (ZipTipMC, Millipore). The method is contingent upon pH-dependent interactions between the phosphate group and immobilized copper(II) ions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with postsource decay confirms the identity and phosphorylation state of the extracted peptide. Peptides containing unphosphorylated histidine residues or other phosphorylated amino acids are not retained, demonstrating the specificity of the method for P-His-containing peptides.
Napper
Scott
S
Department of Biochemistry, Health Sciences Building, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK S7N 5E5 Canada. napper@sask.usask.ca
Kindrachuk
Jason
J
Olson
Douglas J H
DJ
Ambrose
Stephen J
SJ
Dereniwsky
Carmen
C
Ross
Andrew R S
AR
D002413:Cations, Divalent / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D010455:Peptides / Q000032:analysis* / Q000737:chemistry*; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D002413:Cations, Divalent; D010455:Peptides; D006639:Histidine; D003300:Copper