Ultrasonically assisted electrospray ionization for LC/MS determination of nucleosides from a transfer RNA digest

Abstract

The use of an ultrasonic nebulizer to assist electrospray ionization mass spectrometry (ESI-MS) has been described and demonstrated with the analysis of a transfer RNA digest by microcolumn LC. The restricted range of mobile-phase compositions amenable to the electrospray process has traditionally placed a severe limitation on the types of LC applications that can be used with ESI-MS. For this reason, an ultrasonic nebulizer configured for LC has been developed that can generate the fine dispersion of liquid required for ESI-MS from any type of mobile phase. In the case presented here, a transfer RNA was enzymatically digested into its substituent nucleosides, which were then analyzed by microcolumn LC. The required mobile-phase gradient (beginning at 5% methanol) falls outside the solvent range that can be used with conventional electrospray. The ultrasonic nebulizer, however, resolves this problem. The fundamental behavior of the four most common nucleosides (cytidine, adenosine, guanosine, uridine) was studied, and conclusions concerning the effects of solution chemistry were drawn. Specifically, signal from the H+ adducts of these species seems to be strongly dependent on the pKa value. Also, effects from several source operating variables were examined. These included capillary exit voltage, drying and focusing gases, and nebulizer frequency. Performance was found to be consistent over a wide range (0-100% methanol) of mobile-phase compositions. The limit of detection for adenosine injected onto a microcolumn was found to be 100 amol. Finally, nucleosides from as little as 150 fmol of RNA (amount prior to digestion) could be detected.

A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status

“…Identification of cytosine (Cyt) and mCyt was obtained by combined UV detection at 254 nm and MS analysis of the chromatographic peaks eluting after 4.5 Ϯ 0.5 min and 6.5 Ϯ 0.5 min, respectively. The ion peak at m͞z ϭ 112 corresponded to Cyt and the ion peak at m͞z ϭ 126 corresponded to mCyt, confirming what was described by others (38). The ion peak at m͞z ϭ 115 corresponded to the stable isotope used as internal standard.…”

Profiling ribonucleotide modifications at full-transcriptome level: a step toward MS-based epitranscriptomics

“…The ensuing samples are typically resolved by coupling liquid chromatography (Esmans et al 1998;Chan et al 2010;Su et al 2014) or capillary electrophoresis (Apruzzese and Vouros 1998) with MS detection (i.e., LCand CE-MS, respectively), which are meant to provide separation and reduction of chemical background, while avoiding undesirable analyte bias. In previous work, we investigated the merits of direct infusion electrospray ionization (ESI) (Yamashita and Fenn 1984;Banks et al 1994) to perform nucleotide analysis in the absence of front-end chromatographic procedures (Quinn et al 2013). The direct approach was evaluated by using standard samples that contained the canonical ribo-and deoxyribonucleotides with the addition of pseudouridine that constitutes the most abundant variant present in nature (Charette and Gray 2000).…”

Ultrasonication-assisted spray ionization mass spectrometry for the analysis of biomolecules in solution

“…For example, high-energy laser [8], heated gas jets [9], low-temperature plasma [10], and nitrogen gas [11] were successfully employed to desorb analytes at ambient condition. However, post-ionization was generally required for these techniques.Ultrasonically assisted electrospray ionization [12,13] was first demonstrated more than a decade ago, using an ultrasonic transducer to enhance the nebulization efficiency of analytes eluted from a liquid chromatography column and then subjecting them to ESI. A method, so called sonic spray ionization (SSI), for small organics and drugs [14 -17] has been developed, in which a solution from a fused-silica capillary is sprayed with a sonic gas flow coaxial to the capillary.…”

“…Ultrasonically assisted electrospray ionization [12,13] was first demonstrated more than a decade ago, using an ultrasonic transducer to enhance the nebulization efficiency of analytes eluted from a liquid chromatography column and then subjecting them to ESI. A method, so called sonic spray ionization (SSI), for small organics and drugs [14 -17] has been developed, in which a solution from a fused-silica capillary is sprayed with a sonic gas flow coaxial to the capillary.…”