title: Rapid separation of phosphopeptides by microchip electrophoresis-electrospray ionization mass spectrometry.
abstract: 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.
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00014 University of Helsinki, Finland.
authors.forename: Elisa
authors.initials: E
authors.lastname: Ollikainen
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00014 University of Helsinki, Finland.
authors.forename: Ashkan
authors.initials: A
authors.lastname: Bonabi
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00014 University of Helsinki, Finland.
authors.forename: Nina
authors.initials: N
authors.lastname: Nordman
authors.affiliation: Department of Materials Science and Engineering, School of Chemical Technology, Aalto University, Finland, Micronova, Tietotie 3, 02150, Finland.
authors.forename: Ville
authors.initials: V
authors.lastname: Jokinen
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00014 University of Helsinki, Finland; Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki, A. I. Virtasen aukio 1, 00014 University of Helsinki, Finland.
authors.forename: Tapio
authors.initials: T
authors.lastname: Kotiaho
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00014 University of Helsinki, Finland.
authors.forename: Risto
authors.initials: R
authors.lastname: Kostiainen
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki, Viikinkaari 5E, 00014 University of Helsinki, Finland. Electronic address: tiina.sikanen@helsinki.fi.
authors.forename: Tiina
authors.initials: T
authors.lastname: Sikanen
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D046209:Electrophoresis, Microchip; D005449:Fluorenes / Q000737:chemistry; D007202:Indicators and Reagents / Q000737:chemistry; D007536:Isomerism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D010766:Phosphorylation; D011972:Receptor, Insulin / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D005449:Fluorenes; D007202:Indicators and Reagents; D010748:Phosphopeptides; C054007:1-(9-fluorenyl)methyl chloroformate; D011972:Receptor, Insulin
keywords: Electrospray ionization; Mass spectrometry; Microchip electrophoresis; Phosphopeptide analysis