Charge-state reduction with improved signal intensity of oligonucleotides in electrospray ionization mass spectrometry

Abstract

The shift of charge states of oligonucleotide negative ions formed in electrospray ionization mass spectrometry to higher mass-to-charge ratio has been accomplished by addition of organic acids and bases to the solution to be electrosprayed. The use of acetic acid or formic acid combined with piperidine and imidazole effectively reduced charge states. Signal intensity and stability were enhanced greatly when the infused solution contained a high percentage of acetonitrile. In addition, the cocktail that contained imidazole, piperidine, and acetic acid in 80% acetonitrile not only reduced charge states, but also substantially suppressed Na adduction. Several oligonucleotides that varied in base composition and length were investigated, and studies of mixtures showed a significant reduction in spectral complexity.

Accurate characterization of the tyrosine hydroxylase forensic allele 9.3 through development of electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry

“…This repeatability could only be achieved using ethanol precipitation 9,10 in conjunction with microdialysis 7,8 and the addition of organic bases, piperidine and imidazole, to the ESI solution. 17,18 Spectra were obtained for the ds-PCR product (82 bp) with a theoretical average mass of 50,535.89 Da (which exceeds the theoretical ability of a 4.7 Tesla field strength to achieve isotopic resolution, which is dependent on the number of ions in the most abundant isotopic peak 58 ). All spectra were achieved with a PCR product concentration of 0.8 pmol/mL and electrosprayed as described in the experimental section at a flow rate of 300 nL/min with a 200 ms shutter pulse which corresponds to 0.8 fmol of PCR product consumed for the 200 ms time period not accounting for ion losses or trapping efficiencies (data not shown).…”

“…For instrumentation that does not resolve adduction, mass spectral peaks are shifted to higher mass resulting in a systematic error. Microdialysis, 7,8 ethanol precipitation, 9,10 high-performance liquid chromatography, 11 spin columns, 12,13 biomagnetic bead separations, [14][15][16] and the addition of organic modifiers 17,18 are some of the approaches that have been employed to address this critical issue.…”

Analysis of short tandem repeat polymorphisms by electrospray ion trap mass spectrometry

“…Independent of the applied purification method, 25 mM imidazole and 25 mM piperidine in 50% acetonitrile were added to each purified sample prior to infusion into the ESI source. These additives have proven to be advantageous for ESI MS analysis of DNA samples due to the efficient suppression of residual cation adducts (32,50). Figure 1a shows the ESI ion trap mass spectrum of the 81 bp PCR product amplified from an individual homozygous at the equine STR locus HMS3 after application of the novel magnetic bead approach.…”

Fragmentation pathway studies of oligonucleotides in matrix-assisted laser desorption/ionization mass spectrometry by charge tagging and H/D exchange

“…For example, why are similar patterns of fragmentation observed both in positive and negative ion modes [46]? Protonation of a nucleobase in the negative-ion mode is thermodynamically unfavorable because of the high gas-phase acidity of the phosphodiester group (210 -230 kcal/mol for nucleobases versus 315 kcal/mol for phosphodiester group as suggested by Muddiman et al [47]). Alternatively, is fragmentation influenced by the gas-phase behavior of the employed matrix?…”