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

Elisa Ollikainen, Ashkan Bonabi, Nina Nordman, Ville Jokinen, Tapio Kotiaho, Risto Kostiainen, Tiina Sikanen*

*Corresponding author for this work

Research output: Contribution to journalArticleScientificpeer-review

10 Citations (Scopus)


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.

Original languageEnglish
Pages (from-to)249-254
Number of pages6
Publication statusPublished - 1 Apr 2016
MoE publication typeA1 Journal article-refereed


  • Electrospray ionization
  • Mass spectrometry
  • Microchip electrophoresis
  • Phosphopeptide analysis


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