Characterization of DNA oligonucleotides by coupling of capillary zone electrophoresis to electrospray ionization Q-TOF mass spectrometry

A procedure for fast and precise molecular weight, purity, and base composition determination of oligonucleotides (in the size range of 20-120 bases) using capillary zone electrophoresis (CZE) coupled by an electrospray interface (ES) on-line to a quadrupole-inlet time-of-flight mass spectrometer (Q-TOFMS) is described. The detection limit for the 5'-monophosphate nucleotides dAMP and TMP was 30 fmol injected for single MS and 400 fmol for a collision-induced daughter ion spectrum (CID-MS/MS). Injection of 1 pmol was the detection limit for the oligonucleotides, The use of the CZE as an injection interface for oligonucleotides successfully removed metal ion adducts, greatly enhancing the sensitivity and the precision of the mass detection without the need for sample preparation steps. Using electrophoretic separation in an ammonium carbonate buffer system (0.1 M, pH 9.68), these metal adducts were removed and exchanged for volatile ammonium ions. A simple sample stacking technique was developed in order to be able to inject sufficient amounts of sample on the capillary prior to electrophoresis, Using this sample stacking technique, the oligonucleotides could be concentrated up to a factor of 100 on the capillary. Given the high mass precision (+/- 50 ppm) and the high mass resolution (peak widths at half-height of 4 Da), CZE-Q-TOFMS proves to be very useful not only for determination of the purity and the length of oligonucleotides (up to 120 bases) but also for confirmation of the expected base composition, making this technique an extremely useful tool for quality control in the field of oligonucleotides.