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Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides.

ABSTRACT: Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences. Graphical Abstract ?.

SUBMITTER: Basiri B

PROVIDER: S-EPMC5569388 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides.

Basiri Babak B Murph Mandi M MM Bartlett Michael G MG

Journal of the American Society for Mass Spectrometry 20170412 8

Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should ...[more]

PMID: 28405940

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Project description:There has been a recent surge in applications of mass spectrometry (MS) to tissue analysis, particularly lipid-based tissue imaging using ambient ionization techniques. This recent growth highlights the need to examine the effects of sample handling, storage conditions, and experimental protocols on the quality of the data obtained. Variables such as time before freezing after organ removal, storage time at -80 °C, time stored at room temperature, heating, and freeze/thaw cycles were investigated for their effect on the data quality obtained in desorption electrospray ionization (DESI)-MS using mouse brain. In addition, analytical variables such as tissue thickness, drying times, and instrumental conditions were also examined for their impact on DESI-MS data. While no immediate changes were noted in the DESI-MS lipid profiles of the mouse brain tissue after spending 1 h at room temperature when compared to being frozen immediately following removal, minor changes were noted between the tissue samples after 7 months of storage at -80 °C. In tissue sections stored at room temperature, degradation was noted in 24 h by the appearance of fatty acid dimers, which are indicative of high fatty acid concentrations, while in contrast, those sections stored at -80 °C for 7 months showed no significant degradation. Tissue sections were also subjected to up to six freeze/thaw cycles and showed increasing degradation following each cycle. In addition, tissue pieces were subjected to 50 °C temperatures and analyzed at specific time points. In as little as 2 h, degradation was observed in the form of increased fatty acid dimer formation, indicating that enzymatic processes forming free fatty acids were still active in the tissue. We have associated these dimers with high concentrations of free fatty acids present in the tissue during DESI-MS experiments. Analytical variables such as tissue thickness and time left to dry under nitrogen were also investigated, with no change in the resulting profiles at thickness from 10 to 25 μm and with optimal signal obtained after just 20 min in the dessicator. Experimental conditions such as source parameters, spray solvents, and sample surfaces are all shown to impact the quality of the data. Inter-section (relative standard deviation (%RSD), 0.44-7.2%) and intra-sample (%RSD, 4.0-8.0%) reproducibility data show the high quality information DESI-MS provides. Overall, the many variables investigated here showed DESI-MS to be a robust technique, with sample storage conditions having the most effect on the data obtained, and with unacceptable sample degradation occurring during room temperature storage.

Dataset Information

Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides.

Publications

Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides.

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