Analysis of phosphopeptides by capillary electrophoresis and matrix-assisted laser-desorption ionization time-of-flight mass spectrometry

Capillary electrophoresis was used for the qualitative as well as quantitative analysis of phosphopeptides. Three pairs of phosphorylated peptides and non-phosphorylated analogues were analyzed by capillary electrophoresis. Each sequence of phosphopeptides contained one phosphoserine, phosphothreonine or phosphotyrosine residue. The capillary electrophoresis analyses were performed in a fused-silica capillary 50 mu m in internal diameter and 100 cm in length. For detection of analyte peptides, absorbance at 214 and 229 nm wavelength in the UV region was measured. To obtain the best resolution, the concentration of analyte and the pH of the electrolyte buffer were optimized. Matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry was used to confirm the molecular mass of the phosphorylated peptides and nonphosphorylated analogues. For quantitative analysis of the phosphorylated peptides using capillary electrophoresis, the linearity of the response of the absorbance at 214 nm was examined. The response of the absorbance for the phosphorylated peptides was linear; thus this validates the method for quantifying phosphopeptides using capillary electrophoresis. The results of our study show that capillary electrophoresis can be widely applied to analyze and characterize many biologically active phosphopeptides and phosphoproteins.