On-Line Microdialysis Sample Cleanup for Electrospray Ionization Mass Spectrometry of Nucleic Acid Samples

Abstract

A major limitation of electrospray ionization mass spectrometry (ESI-MS) for oligonucleotide analysis arises due to sodium adduction, a problem that increases with molecular weight. Sodium adduction can preclude useful measurements when limited sample sizes prevent off-line cleanup. A novel and generally useful on-line microdialysis technique is described for the rapid (∼1-5 min) DNA sample cleanup for ESI-MS. Mass spectra of oligonucleotides of different size and sequence showing no significant sodium adduct peaks were obtained using the on-line microdialysis system with sodium chloride concentrations as high as 250 mM. Signal-to-noise ratios were also greatly enhanced compared to direct infusion of the original samples. By using ammonium acetate as the dialysis buffer, it was also found that the noncovalent association of double-stranded oligonucleotides could be preserved during the microdialysis process, allowing analysis by ESI-MS.

The observation of chaperone‐ligand noncovalent complexes with electrospray ionization mass spectrometry

“…The approach that we have employed for the present work is to unfold the ligand protein with the use the denaturant GuHCl (3 M), rapidly dilute the denatured protein away from the denaturant in the presence of SecB to allow interaction, and then dialyze the solution to remove the high salt concentration immediately prior to mass spectral analysis. In addition, to slow the refolding of the OppA and prolong the existence of the complex, the rapid microdialysis step (Liu et al, 1996) of SecB-OppA complexes was carried out at 0°C. The interaction of SecB with its ligands is characterized by a rapid equilibrium between the bound and free states.…”

“…Reaction mixtures were then prepared using the denatured OppA solution and stock solutions of SecB (145 micromolar) and DTT (10 mM), such that the final concentrations of SecB and OppA were I O and 18 micromolar, respectively. The final concentration of GuHCl was 0.2 M. Immediately after mixing, samples were dialyzed against a solution of 10 mM NH,OAc and 10 mM DTT at O"C, as mentioned above, and then analyzed immediately by ESI-FTICR-MS. With the microdialysis setup employed (Liu et al, 1996) dialysis of reaction mixtures was carried out in 2-4 min.…”

A eole for the MS analysis of nucleic acids in the post-genomics age

“…Indeed, the negative charges afforded by these functional groups confer nucleic acids a strong tendency to form unwanted cation adducts that can deteriorate the attainable resolution and signal to noise ratio. Over the years, alternative strategies have been devised to replace metal cations with the more volatile ammonium [42,177], which employ ion-exchange [178], reversed-phase high-performance liquid chromatography [179], metal chelation [180,181], ethanol precipitation [177], ultrafiltration, and microdialysis [182][183][184]. Although separationbased desalting is very effective for these types of biomolecules, it may also result in the unwanted dissociation of their noncovalent assemblies, either through excessive reduction of the solution ionic strength, or through direct perturbation of binding equilibria.…”

Solution composition and thermal denaturation for the production of single-stranded PCR amplicons: Piperidine-induced destabilization of the DNA duplex?

“…All oligonucleotides were desalted prior to ESI using the microdialysis apparatus previously described [18,39] except with a molecular weight cut off (MWCO) of 18,000 Da for the dialysis membrane. Concentrations for the stock solutions of oligonucleotides were quantified using UV-Vis spectrophotometry at 260 nm prior to dilution.…”