Quantification of mRNA cap-modifications by means of LC-QqQ-MS

Muthmann, Nils; Špaček, Petr; Reichert, Dennis; Dülmen, Melissa van; Rentmeister, Andrea

doi:10.1016/j.ymeth.2021.05.018

Cited by 13 publications

(18 citation statements)

References 52 publications

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“…2a-c, Supplementary Fig. 1-2) 58 . This analysis showed that the level of propargylated A (A prop /A) was increased at higher (up to 2.5 mM) PSH concentrations (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Digested, dephosphorylated nucleoside mix was separated with a linear gradient from buffer A (20 mM NH 4 OAc in ddH 2 O (pH 6.0) to buffer B (acetonitrile) (see Supplementary Table 6) at 40 °C with 0.8 mL/min. The principle of measuring, optimizing mass spectrometric parameters, and quantifying modified nucleosides was described before in great detail by Muthmann et al 58 . For best performance, mass spectrometric parameters such as fragmentor voltage (FV) and collision energy (CE) were optimized individually for each product ion.…”

Section: Methodsmentioning

confidence: 99%

“…of all analytes and respective RET windows are listed in Supplementary Table 7. For quantification, pairs of precursor ion and product ion were detected, using the most abundant MRM transition - fragment of ribose loss or ribose derivative loss 58 - as quantifier. The second most abundant or specific MRM transition was used as a qualifier and the ratio between quantifier and qualifier signals was used as extra evidence for analyte identity.…”

Section: Methodsmentioning

confidence: 99%

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MePMe-seq: Antibody-free simultaneous m⁶A and m⁵C mapping in mRNA by metabolic propargyl labeling and sequencing

Hartstock

Ovcharenko

Kueck

et al. 2022

Preprint

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Internal modifications of mRNA have emerged as widespread and versatile regulatory mechanism to control gene expression at the post-transcriptional level. Current insights rely on the ability to make a modified nucleoside amenable to sequencing. Most of the modifications are methylations involving the co-factor S-adenosyl-L-methionine (SAM), however, simultaneous detection of different methylation sites in the same sample has remained elusive. We present metabolic labeling with propargyl-selenohomocysteine (PSH) in combination with click chemistry to detect N6-methyladenosine (m6A) and 5-methylcytidine (m5C) sites in mRNA with single nucleotide precision in the same sequencing run (MePMe-seq). Intracellular formation of the corresponding SAM analogue leads to detectable levels of N6-propargyl-A (prop6A) and 5-propargyl-C (prop5C). MePMe-seq overcomes the problems of antibodies for enrichment and sequence-motifs for evaluation, limiting previous methodologies. The joint evaluation of m6A and m5C sites opens the door to study their interconnectivity and improve our understanding of mechanisms and functions of the RNA methylome.

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“…2a-c, Supplementary Fig. 1-2) 58 . This analysis showed that the level of propargylated A (A prop /A) was increased at higher (up to 2.5 mM) PSH concentrations (Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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MePMe-seq: Antibody-free simultaneous m⁶A and m⁵C mapping in mRNA by metabolic propargyl labeling and sequencing

Hartstock

Ovcharenko

Kueck

et al. 2022

Preprint

Self Cite

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“…In a different approach, Muthmann and co-authors digested and dephosphorylated their entire RNA transcript based on an incubation with snake venom nuclease P1. From this, they obtained single nucleosides and were able to use gel electrophoresis and MS to quantify different cap species [81] , [82] . For industry laboratories, it might remain desirable to apply an RNase H based sample preparation because reagents are readily available.…”

Section: ’ Endcap Analysismentioning

confidence: 99%

Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals

Fekete¹,

Doneanu

Addepalli

et al. 2023

Journal of Pharmaceutical and Biomedical Analysis

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“…In addition, in 2021 Muthmann and co-authors performed digestion of the entire transcript as initiated by nuclease P1, snake venom phosphodiesterase, and dephosphorylation. Finally, they generated single nucleosides with or without the cap and quantified these products with triple quadrupole mass spectrometry [ 91 ].…”

Section: Analytical Approachesmentioning

confidence: 99%

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

et al. 2021

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In the quest for a formidable weapon against the SARS-CoV-2 pandemic, mRNA therapeutics have stolen the spotlight. mRNA vaccines are a prime example of the benefits of mRNA approaches towards a broad array of clinical entities and druggable targets. Amongst these benefits is the rapid cycle “from design to production” of an mRNA product compared to their peptide counterparts, the mutability of the production line should another target be chosen, the side-stepping of safety issues posed by DNA therapeutics being permanently integrated into the transfected cell’s genome and the controlled precision over the translated peptides. Furthermore, mRNA applications are versatile: apart from vaccines it can be used as a replacement therapy, even to create chimeric antigen receptor T-cells or reprogram somatic cells. Still, the sudden global demand for mRNA has highlighted the shortcomings in its industrial production as well as its formulation, efficacy and applicability. Continuous, smart mRNA manufacturing 4.0 technologies have been recently proposed to address such challenges. In this work, we examine the lab and upscaled production of mRNA therapeutics, the mRNA modifications proposed that increase its efficacy and lower its immunogenicity, the vectors available for delivery and the stability considerations concerning long-term storage.

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Quantification of mRNA cap-modifications by means of LC-QqQ-MS

Cited by 13 publications

References 52 publications

MePMe-seq: Antibody-free simultaneous m⁶A and m⁵C mapping in mRNA by metabolic propargyl labeling and sequencing

MePMe-seq: Antibody-free simultaneous m⁶A and m⁵C mapping in mRNA by metabolic propargyl labeling and sequencing

Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

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Quantification of mRNA cap-modifications by means of LC-QqQ-MS

Cited by 13 publications

References 52 publications

MePMe-seq: Antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing

MePMe-seq: Antibody-free simultaneous m6A and m5C mapping in mRNA by metabolic propargyl labeling and sequencing

Challenges and emerging trends in liquid chromatography-based analyses of mRNA pharmaceuticals

mRNA Therapeutic Modalities Design, Formulation and Manufacturing under Pharma 4.0 Principles

Contact Info

Product

Resources

About

MePMe-seq: Antibody-free simultaneous m⁶A and m⁵C mapping in mRNA by metabolic propargyl labeling and sequencing

MePMe-seq: Antibody-free simultaneous m⁶A and m⁵C mapping in mRNA by metabolic propargyl labeling and sequencing