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Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays.

ABSTRACT: Mass spectrometry is a widely used tool in the characterization of oligonucleotides. This analysis can be challenging due to the large number of possible charge states of oligonucleotides, which can limit the sensitivity of the assay, along with the propensity of oligonucleotides to readily form adducts with free alkali metals. To reduce the adduct formation, oligonucleotides are typically purified with desalting columns prior to analysis. We have developed a mobile phase that gives superior reduction in charge states and adduct formation compared to previously reported methods and, more importantly, obviates the requirement of desalting samples prior to mass spectrometric analysis, significantly decreasing the sample preparation time and amount of RNA required for analysis. We have applied this mobile phase to develop methods to quantify the 5'-capping efficiency and to characterize the polyadenosine (poly(A)) tail of mRNA synthesized in vitro: two critical quality attributes of mRNA therapeutics. Through this, we were able to demonstrate RNA that was co-transcriptionally capped to have capping efficiency equivalent (the percent total molecules that contain a cap) to other reports in the literature using materials that were generated using the same synthesis procedure. Furthermore, by using a mobile phase mixture comprised of hexafluoroisopropanol, triethylammonium acetate, triethylamine, and ethanol, we were able to determine the size distribution of the poly(A) tail in various mRNA samples from DNA templates that ranged from 50 to 150 nt poly(A) and verify that distribution with commercially available RNA standards, successfully demonstrating that this mobile phase composition could be used for characterization assays for both mRNA caps and tails.

SUBMITTER: Strezsak SR

PROVIDER: S-EPMC9260895 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Diacylglycerols (DAGs) are important lipid mediators in cellular signaling transduction and metabolism. Imbalanced production or consumption of DAGs has a negative impact on the physiological functions of the body. However, comprehensive monitoring of structurally diverse DAGs remains a daunting task due to the rapid metabolism and ion suppression characteristics in biofluids. These bottlenecks call for developing a method that enables sensitive quantification of DAGs in biological sample. In this work, a straightforward charge derivatization strategy was developed to insert a series of structure analogs charge label, i.e., N, N-dimethylglycine (DMG) and N, N-dimethylalanine (DMA), on the free hydroxyl group of the DAGs. Owing to the existence of tertiary amino groups in charge label, the mass spectrometry ionization response of the derivatized DAGs was significantly increased in comparison with traditional metal ion adducts. After charge derivatization, the specific neutral loss diagnostic ions (DMG, 103 Da and DMA, 117 Da) were captured by mass spectrometry. Then, the DMG/DMA-oriented paired multiple reaction monitoring methods based on the characteristic diagnostic ions of the derivatized DAGs have been developed as sensitive methods for the detection (detection limit = 16 aM) and quantification (quantification limit = 62.5 aM) of DAGs in serum. Moreover, the tagged 1,2-DAGs and 1,3-DAGs sn-isomers have been well separated on the reversed-phase column in combination with ultra-performance liquid chromatography. Finally, metabolic characterizations of the tagged DAGs were further explored in L-Arginine-induced acute pancreatitis mice and resveratrol treated model mice. The results indicated that 1,2-DAGs were increased in the serum of model mice relative to normal controls and resveratrol significantly altered this metabolic abnormality. The currently established DMG/DMA-oriented paired charge derivatization strategy is promising for depicting DAGs changes more accurately in metabolic studies of lipid-related diseases and accurately evaluating drug treatment strategies.

Project description:Alkali and alkaline earth metal adducts of a branched glycan, XXXG, were analyzed with helium charge transfer dissociation (He-CTD) and low-energy collision-induced dissociation (LE-CID) to investigate if metalation would impact the type of fragments generated and the structural characterization of the analyte. The studied adducts included 1+ and 2+ precursors involving one or more of the cations: H+ , Na+ , K+ , Ca2+ , and Mg2+ . Regardless of the metal adduct, He-CTD generated abundant and numerous glycosidic and cross-ring cleavages that were structurally informative and able to identify the 1,4-linkage and 1,6-branching patterns. In contrast, the LE-CID spectra mainly contained glycosidic cleavages, consecutive fragments, and numerous neutral losses, which complicated spectral interpretation. LE-CID of [M + K + H]2+ and [M + Na]+ precursors generated a few cross-ring cleavages, but they were not sufficient to identify the 1,4-linkage and 1,6-branching pattern of the XXXG xyloglucan. He-CTD predominantly generated 1+ fragments from 1+ precursors and 2+ product ions from 2+ precursors, although both LE-CID and He-CTD were able to generate 1+ product ions from 2+ adducts of magnesium and calcium. The singly charged fragments derive from the loss of H+ from the metalated product ions and the formation of a protonated complementary product ion; such observations are similar to previous reports for magnesium and calcium salts undergoing electron capture dissociation (ECD) activation. However, during He-CTD, the [M + Mg]2+ precursor generated more singly charged product ions than [M + Ca]2+ , either because Mg has a higher second ionization potential than Ca or because of conformational differences and the locations of the charging adducts during fragmentation. He-CTD of the [M + 2Na]2+ and the [M + 2 K]2+ precursors generated singly charged product ions from the loss of a sodium ion and potassium ion, respectively. In summary, although the metal ions influence the mass and charge state of the observed product ions, the metal ions had a negligible effect on the types of cross-ring cleavages observed.

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Novel Mobile Phase to Control Charge States and Metal Adducts in the LC/MS for mRNA Characterization Assays.

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