On-line coupling of capillary gel electrophoresis with electrospray mass spectrometry for oligonucleotide analysis.
Homooligodeoxyribonucleotides differing one nucleotide in length from 12- to 15-mer and from 17- to 20-mer were separated by size with capillary gel electrophoresis (CGE) using an entangled polymer solution in coated capillaries. The resolved components were analyzed by on-line coupling of CGE with electrospray mass spectrometry (ES-MS), denoted as CGE/ES-MS, in the full-scan negative ion detection mode. Baseline separation was achieved for the 12-15-mer oligonucleotide mixtures. Both synthetic phosphodiester oligonucleotide mixtures as well as their phosphorothioate analogues, serving as model compounds for antisense oligonucleotides, could be analyzed by on-line CGE/ES-MS coupling. Terminally phosphorylated and nonphosphorylated synthetic failure sequences could be electrophoretically separated and mass spectrometically characterized as well. This methodology might be a useful tool for synthesis control of phosphodiester oligonucleotides as well as for analysis of phosphorothioate analogues as they are used in antisense drug development.
Freudemann
T
T
Research Center for Nucleic Acid and Peptide Chemistry, Institute for Organic Chemistry, University of Tübingen, Germany.
von Brocke
A
A
Bayer
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E
D019075:Electrophoresis, Capillary / Q000379:methods*; D009841:Oligonucleotides / Q000032:analysis*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D009841:Oligonucleotides