Data for: Oligonucleotide mapping via mass spectrometry to enable comprehensive primary structure characterization of an mRNA vaccine against SARS-CoV-2
Description
Oligonucleotide mapping via liquid chromatography mass spectrometry mass spectrometry (LC-MS/MS) was recently developed to support development of Comirnaty®, the world's first commercial mRNA vaccine which immunizes against the SARS-CoV-2 virus. Analogous to peptide mapping of therapeutic protein modalities, oligonucleotide mapping described here provides direct primary structure characterization of mRNA, through enzymatic digestion, accurate mass determinations, and optimized collisionally-induced fragmentation. Sample preparation for oligonucleotide mapping is a rapid, one-pot, one-enzyme digestion. The digest is analyzed via LC-MS/MS with an extended gradient and resulting data analysis employs semi-automated software. In a single method, oligonucleotide mapping readouts include a highly reproducible and completely annotated UV chromatogram with >98% sequence coverage and a microheterogeneity assessment of 5´ terminus capping and 3´ terminus poly(A) tail length. Oligonucleotide mapping was pivotal to ensure the quality, safety, and efficacy of mRNA vaccines by providing: confirmation of construct identity and primary structure and assessment of product comparability following manufacturing process changes. More broadly, this technique may be used to directly interrogate the primary structure of RNA molecules in general.
Notes
The Thermo mass spectrometer .raw files can be opened by Xcalibur Qual Browser (Thermo Fisher Scientific), which is (proprietary) commercial software. An open-source alternative is ThermoRawFileParser (https://doi.org/10.1021%2Facs.jproteome.9b00328).
Automated identification of oligonucleotides was accomplished using BioPharma Finder v5.0 (Thermo Fisher Scientific), which is (propriety) commercial software. An open-source alternative is attached in this dataset: three Excel VBA spreadsheets that may be used to in the identification of any MS feature. As Excel VBA spreadsheets, they are fully compiled and do not require any library code extensions or external links.
The "Oligonucleotide MS Peak ID Given Sequence v8.xlsm" spreadsheet may be used to generate a list of theoretical RNaseT1 digest oligonucleotides and match observed precursor masses to candidate theoretical RNaseT1 digest oligonucleotides. Inputs are a single or list of observed masses and a target mRNA construct sequence.
The "Oligonucleotide MS2 Spectrum Matcher v11.xlsm" spreadsheet may be used to check a candidate oligonucleotide sequence with a single MS/MS spectrum. Inputs are the candidate sequence and the m/z vs intensity "XY" coordinate list.
The "Oligonucleotide Composition from Mass Calculator v2.xlsm" may be used to generate a list of possible nucleotide compositions given an input mass.
A 4th Excel VBA spreadsheet is also provided, "Oligonucleotide Mapping UV Annotation Tool v11.xlsm", to facilitate the annotation of the LC-UV chromatogram with LC-MS/MS-identified oligonucleotides in a PowerPoint file in which the chromatogram is plotted with annotations placed as separate, overlayed text objects in the .pptx. Inputs to this spreadsheet include a Qual Browser LC/UV peak list, LC/UV chromatogram time vs intensity (XY) coordinate list, the mRNA construct sequence, and the BioPharma Finder-exported component table.
The use of these spreadsheets and details of the entire method are provided in Oligonucleotide Mapping and Data Analysis Protocol.pdf
Funding provided by: Pfizer
Crossref Funder Registry ID: http://dx.doi.org/10.13039/100004319
Award Number:
Files
GAUB_DAWDYA_17MAR2023_OligonucleotideMappingMethod_ProtocolExchange.pdf
Files
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