Quantification of dsRNA using stable isotope labeling dilution liquid chromatography/mass spectrometry

Kung, An-Wen; Kilby, Peter M.; Portwood, David E.; Dickman, Mark J.

doi:10.1002/rcm.8074

Cited by 7 publications

(3 citation statements)

References 27 publications

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“…The use of mass spectrometry, on the one hand, provides the opportunity to ensure the identity of the molecule but requires careful sample preparation, and on the other hand, avoids matrix effects and ion suppression [19]. A commonly used procedure is the use of stable isotope-labeled internal standards to quantify the compound of interest [20,21]. However, a gap exists when it comes to quantifying oligonucleotides using mass spectrometry compared to protein and peptides.…”

Section: Introductionmentioning

confidence: 99%

Quantification of Oligonucleotides Using Tandem Mass Spectrometry with Isobaric Internal Standards

Gawlig,

Hanci,

Rühl

2023

IJMS

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In recent years, oligonucleotides have become more important in research, drug approvals and medical therapies. Due to this growing interest in pharmaceutical applications, it is essential to develop reliable analytical methods for this substance class. In this work, we present a quantification method using liquid chromatography coupled with tandem mass spectrometry by applying an isobaric oligonucleotide standard. In addition to a proof of principle, we perform a method qualification to assess its readiness for validation according to ICH Q2 guidelines. In addition to good linearity, sensitivity, accuracy and recovery, the method showed no significant matrix effects. Furthermore, we demonstrated the application of the method by applying the quantification in a biological matrix, as well as an exemplary degradation of an oligonucleotide in bovine plasma.

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Section: Introductionmentioning

confidence: 99%

Quantification of Oligonucleotides Using Tandem Mass Spectrometry with Isobaric Internal Standards

Gawlig,

Hanci,

Rühl

2023

IJMS

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“…However, there are some parallels between the analysis of phosphorothioate oligonucleotides and proteins or RNA in complex mixtures. Stable isotope labeling methods have been used successfully for quantitating proteins and RNA by mass spectrometry in a variety of matrices. A similar approach could be applied for measuring assay in phosphorothioate oligonucleotides, but this would require a heavy isotope reference standard for each compound in development, which would be a considerable drawback considering the regulatory requirements for generation, qualification, and maintenance of reference standards following good manufacturing practices.…”

Section: Introductionmentioning

confidence: 99%

Assay determination by mass spectrometry for oligonucleotide therapeutics

Madsen

Roussis

Schniepp

et al. 2019

Rapid Comm Mass Spectrometry

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Phosphorothioate oligonucleotide drugs typically contain product-related impurities that are difficult to resolve chromatographically from the parent oligonucleotide due to the size of these compounds and the large number of stereoisomers that comprise the parent. The presence of co-eluting impurities hinders the process of determining assay based on chromatographic separation alone. A mass spectrometry-based purity assessment of the main chromatography peak can be used to quantify co-eluting impurities and enable the accurate determination of assay, but a more direct measure of assay was desired due to the complexity of measuring all co-eluting impurities by mass spectrometry. Therefore, we developed an assay method that utilizes the specificity of mass spectrometry to measure the amount of active pharmaceutical ingredient in a sample, which eliminates the need for chromatographic separation of impurities from the product. This procedure uses a single quadrupole mass spectrometer and incorporates an internal standard that is co-sprayed with the analyte to compensate for the drift commonly associated with mass spectrometry-based quantitation. Using the mass spectrometry response ratio for sample to internal standard enables the method to achieve excellent linearity (R 2 = 0.998), repeatability (relative standard deviation = 0.5%), intermediate precision (0.6%), and accuracy, with measured assay values consistently within 2.0% of expected. The results indicate the method possesses the accuracy and precision required for measuring assay in clinical and commercial stage pharmaceutical products. Since the method is based on the specificity of the mass spectrometer, and does not rely on chromatographic separation of impurities, the procedure should be applicable to a wide variety of oligonucleotide therapeutics regardless of sequence or chemical modifications.

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“…85 Quantitative analysis of dsRNA is also possible by using metabolically prepared RNA form E. coli. 221 Specifically, for absolute quantification of nucleosides, stable isotope labeled compounds are necessary to overcome the limitations of MS. In quantification, the signal intensity of an analyte must correlate with its concentration or amount of analyte, but in addition on a multitude of other parameters such as salt load, ionization properties of the analyte, instrument parameters and so on.…”

Section: Isotope Labeling Of Biomolecules As a Tool For Analysismentioning

confidence: 99%

Mass spectrometric studies of RNA modification patterns in mammals

Atoi¹

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RNAs are key players in life as they connect the genetic code (DNA) with all cellular processes dominated by proteins. The dynamics study of RNA modifications has become an important part of epitranscriptomics field, as they are reversible and dynamically regulated far more than originally thought. Several evidences portrait a catalog of RNA modifications and their links to neurological disorders, cancers, and other diseases. Therefore, a deeper investigation of RNA modifications dynamics including their specific profile, biosynthesis, maturation and degradation is required for pioneering disease diagnostics and potential therapeutics development. Mammalian tissues reveal diverse physiology and functions, despite sharing identical genomes and overlapping transcription profiles. So far, most research on this diversity were referred to variable transcriptomic processing among tissues and differential post-translational modifications that tune the activity of ubiquitous proteins to each tissue’s needs. However, study of epitranscriptome dynamics relevance to tissues’ functions is not yet revealed. There are a few reports on mouse RNA modification profiles, which are focused on only one type of RNA and limited types of modifications. The first part of my dissertation aims to generate a comprehensive tissue-specific as well as RNA species-specific investigation of all existing RNA modifications, as well as investigating potential codon as an effector of translation diversity among tissues. Using isotope dilution mass spectrometry, I created a library including absolute quantification of 24 tRNA modifications, and up to 22 rRNA modifications. I find an almost identical pattern of modifications in 28S- and 18S-rRNA subunits, but different levels of most modifications in 5.8S-rRNA or tRNA among highly metabolic active organs to e.g. heart or spleen. The findings suggest a high degree of similarity between quantities of modifications between presented data to all previous literature, confirming that it is a suitable model to study the tissue-based RNA modification patterns. The most noticeable difference exhibited was tRNA modifications, which suggests a discerning tRNA engagement in translation between different organs. This can be a good start for investigation of codon bias in enriched genes of specific tRNA modifications among different tissues that may cause differential translation pattern, causing organs diversity. Moreover, 5.8S rRNA data showed an organ-specific pattern, which proposes functional diversity of this rRNA subunit among different organs. Future studies must investigate the possible implications of organ-specific 5.8S rRNA modifications functions, to elucidate the core of the observed variations. Abundance of RNA modifications is carefully regulated in cells. Part of this regulation is achieved by activity of enzymes removing RNA modifications, named RNA erasers. Literature has provided proof of demethylation activity of AlkBH family on different types of RNA. For instance, AlkBH5 is known to remove m6A in mRNA, and both AlkBH3 and AlkBH1 are reported to demethylate m1A and m3C in tRNA. So far, RNA erasers are mainly studied in vitro and direct in vivo studies are missing. Mass spectrometry is a promising approach in the identification and quantification of many RNA modifications. However, mass spectrometric analysis by nature, offers only a static view of nucleic acid modifications, and fails to account for their cellular dynamics. Nucleic Acid Isotope Labeling coupled Mass Spectrometry (NAIL-MS) was developed as a powerful technique which differentiates among remaining, co-transcriptional and post-transcriptional incorporation of a target RNA modification. This temporal resolution captures the dynamic nature of RNA modifications, and offers absolute and relative quantification of all existing nucleosides in any given RNA sequence, including different isotopologues and isotopomers. The objective of this study was to uncover the first “direct” iv vivo data on AlkBH1, 3 and 5 activities in demethylating each of their specific substrates. I investigated the RNA modification changes through pulse-chase experiments in collaboration with my colleagues Dr. Kayla Borland and Dr. Felix Hagelskamp. A remarkable observation was that AlkBH3 protein -but not AlkBH1- was overexpressed under methylating reagent treatment in vivo. These findings suggest that AlkBH3 -but not AlkBH1- is a methylation damage induced enzyme, that potentially triggers ASCC-AlkBH3 alkylation repair complex after aberrant methylation damage by MMS treatment. However, using NAIL-MS method, we could not detect any significant effect on demethylation activity of the enzymes in tRNA, rRNA or mRNA towards the possible substrates m6A, m1A, m3C, m5C and m7G in vivo. These distinct outcomes can be partially explained by probable existence of other unidentified demethylases that compensate for AlkBHs demethylation activity; or more probably, demethylation may still arise by remaining active AlkBHs to restore the original levels of the observed RNA modifications, since a stronger KD or a complete knockout of AlkBHs genes was not possible. Further research on fully knocked out AlkBHs genes can provide stronger evidence on unidentified demethylation activities in HEK cells.

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Quantification of dsRNA using stable isotope labeling dilution liquid chromatography/mass spectrometry

Abstract: Stable isotope labeling of dsRNA in conjunction with MS enabled the characterisation and quantification of dsRNA in complex total RNA mixtures.

Cited by 7 publications

References 27 publications

Quantification of Oligonucleotides Using Tandem Mass Spectrometry with Isobaric Internal Standards

Quantification of Oligonucleotides Using Tandem Mass Spectrometry with Isobaric Internal Standards

Assay determination by mass spectrometry for oligonucleotide therapeutics

Mass spectrometric studies of RNA modification patterns in mammals

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