Rapid separation of phosphopeptides by microchip electrophoresis-electrospray ionization mass spectrometry.

Protein phosphorylation is a significant biological process, but separation of phosphorylated peptide isomers is often challenging for many analytical techniques. We developed a microchip electrophoresis (MCE) method for rapid separation of phosphopeptides with on-chip electrospray ionization (ESI) facilitating online sample introduction to the mass spectrometer (MS). With the method, two monophosphorylated positional isomers of insulin receptor peptide (IR1A and IR1B) and a triply phosphorylated insulin receptor peptide (IR3), all with the same amino acid sequence, were separated from the nonphosphorylated peptide (IR0) in less than one minute. For efficient separation of the positional peptide isomers from each other derivatization with 9-fluorenylmethyl reagents (either chloroformate, Fmoc-Cl, or N-succinimidyl carbonate, Fmoc-OSu) was required before the analysis. The derivatization improved not only the separation of the monophosphorylated positional peptide isomers in MCE, but also identification of the phosphorylation site based on MS/MS.