Protocol

Capillary Electrophoresis

Volume 384 of the series Methods In Molecular Biology™ pp 401-414

Analysis of Oligonucleotides Using Capillary Zone Electrophoresis and Electrospray Mass Spectrometry

  • An WillemsAffiliated withLaboratory of Medical Biochemisty and Clinical Analysis, Ghent University
  • , Dieter L. DeforceAffiliated withLaboratory of Medical Biochemisty and Clinical Analysis, Ghent University
  • , Jan Van BocxlaerAffiliated withLaboratory of Medical Biochemisty and Clinical Analysis, Ghent University

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Summary

This chapter illustrates the usefulness of capillary zone electrophoresis (CZE) coupled to high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry for the single-step desalting, and separation, as well as characterization of oligonucleotides in the framework of quality control after synthesis. Separation is performed using a 25 mM ammonium carbonate buffer supplemented with 0.2 mM trans-1,2-diaminocyclohexane-N, N, N′, N′ id (CDTA) (pH 9.7). During the electrophoretic process, sodium and potassium ions are removed from the polyanionic backbone of the oligonucleotides by exchange of these ions with ammonium ions or by chelation on CDTA, thus eliminating a sample preparation step. A sample stacking procedure used to concentrate the samples on the CZE capillary is described. After analysis, the obtained spectrum is deconvoluted to the zero charge spectrum to yield the molecular mass of the oligonucleotide. A misincorporation of one nucleotide can be detected by a difference in mass.

Keywords

Capillary zone electrophoresis electrospray mass spectrometry quadrupole time-of-flight mass spectrometer oligonucleotides quality control