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 co mposition determination of oligonucleotides (in the size range of 20-1 20 bases) using capillary zone electrophoresis (CZE) coupled by an ele ctrospray 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 sin gle MS and 400 fmol for a collision-induced daughter ion spectrum (CID -MS/MS). Injection of 1 pmol was the detection limit for the oligonucl eotides, The use of the CZE as an injection interface for oligonucleot ides successfully removed metal ion adducts, greatly enhancing the sen sitivity and the precision of the mass detection without the need for sample preparation steps. Using electrophoretic separation in an ammon ium carbonate buffer system (0.1 M, pH 9.68), these metal adducts were removed and exchanged for volatile ammonium ions. A simple sample sta cking technique was developed in order to be able to inject sufficient amounts of sample on the capillary prior to electrophoresis, Using th is sample stacking technique, the oligonucleotides could be concentrat ed up to a factor of 100 on the capillary. Given the high mass precisi on (+/- 50 ppm) and the high mass resolution (peak widths at half-heig ht of 4 Da), CZE-Q-TOFMS proves to be very useful not only for determi nation of the purity and the length of oligonucleotides (up to 120 bas es) but also for confirmation of the expected base composition, making this technique an extremely useful tool for quality control in the fi eld of oligonucleotides.