Modeling cationic adduction of oligonucleotides using electrospray desorption ionization

Abstract

Rationale Cationic adduction causes poor sensitivity and increases spectral complexity during mass spectral analysis of oligonucleotides and alkylamines are used to reduce this adduction. It is unclear the effect of the physiochemical properties of the alkylamines on the reduction of the cationic adduction. Methods All samples were directly infused into a Synapt G2 HDMS quadrupole time‐of‐flight (TOF) hybrid mass spectrometer in negative ion electrospray ionization mode through the native built‐in fluidics system. The infusion flow rate was set to 50 μL/min. The TOFMS tuning parameters were as follows: capillary voltage −2.0 kV, cone voltage 25 V, extraction cone voltage 2 V, source temperature 125°C, desolvation temperature 450°C, cone gas flow rate 0 L/h, and desolvation gas (nitrogen) flow rate 1000 L/h. Results A quantitative model was created to predict the optimized alkylamine for MS analysis, while a qualitative model was generated to explain the most important physiochemical properties: proton affinity (13.83%), gas‐phase basicity (11.79%), pKa (11.47%), boiling point (10.73%), MW (10.3%), Henry's Law Constant (9.56%), and partition coefficient (logP) (9.44%). The quantitative model was applied to RNA (microRNA) and a phosphorothioate and predicts the trend of cationic adduction. Conclusions Two models are described to understand the physiochemical properties that contribute to the adduction and to provide users a quick mathematical tool to predict the best choice of alkylamine to lower cationic adduction and decrease spectral complexity while enhancing sensitivity.

Oligonucleotide Anion Adduct Formation Using Negative Ion Electrospray Ion-Mobility Mass Spectrometry

“…Anionic salt solutions were freshly made at 1 mg/mL. The final concentrations of the working solution in 50:50 methanol/water with 15 mM octylamine (OA) and 25 mM fluorinated alcohol, except for NFTB, which was used at 2 mM, were 10 μM oligonucleotide and 75 μM anionic salt. , All surfaces and equipment were cleaned with 1% sodium dodecyl sulfate followed by 70% ethanol prior to analysis to prevent degradation of the oligonucleotide samples. Eppendorf DNA Lobind tubes (Hauppauge, NY) were used to minimize any nonspecific loss of the oligonucleotides to plastic surfaces.…”

“…Oligonucleotides have a highly acidic backbone and are generally analyzed in negative ion mode with electrospray. Cations can highly adduct to the phosphate backbone and cause poor sensitivity by causing the signal intensity to be spread across many charges and adduct states . However, anionic adducts have been shown to provide stability and can be used in mass spectrometry experiments to provide unique data. − Fabrik et al leveraged phosphate anions to study the structural features of free oligosaccharides .…”

Massively Parallel Selection of NanoCluster Beacons

“…Since our NCB constructs were much larger (45-nt long for the NC probe strand and 48-nt long for the activator strand) than the constructs used in previous studies involving ESI-MS analysis 10,38,39 (10to 26-nt-long), extensive cationic metal adduction during the ESI process prevented us from deciphering the exact silver stoichiometries in purified NCB samples. To circumvent this issue, an ionpairing reagent, octylamine, was added to the ESI samples to suppress salt adduct formation [40][41][42][43] .…”

“…Two gel-purified NCB samples (yPOT5 and rPOT5) were desalted and buffer exchanged into 10 mM ammonium acetate using a spin column (Micro Bio-Spin™ P-6 Gel Columns, Bio-Rad). Octylamine was added to aliquots of NCB solution at a concentration of 0.1% (v/v), to reduce the extensive metal cationic adduction that is commonly seen for ESI-MS analysis of oligonucleotides > 20 nt [40][41][42][43] .…”

Bioanalysis and Biotransformation of Oligonucleotide Therapeutics by Liquid Chromatography‐mass Spectrometry

“…Oligonucleotides suffer from poor sensitivity due to being analyzed in the negative ion mode, due to the negative ion mode causing an envelope of charges and an additional envelope of cationic adducts (Muddiman et al, 1996; Sutton & Bartlett, 2019). In addition, most of these therapeutic oligonucleotides are dosed infrequently (2–3 weeks to months apart) due to their relatively long half‐lives.…”