Modeling MS1 and MS2 Effectiveness for mRNA Sequence Confirmation

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Mass spectrometry (MS) offers a single-instrument method for verification of mRNA vaccines including primary sequence, 5’ cap, poly-A tail, and modifications. Due to their size, mRNAs must be enzymatically digested for MS analysis. See Gau, Dawdy, et al., 2023, PMID 37270636. Challenges include:

• Size of mRNAs. ~4300 nucleotides and ~1.3 MDa for Covid-19 vaccines
• Duplicate digestion pieces. ACAAG appears three times as a fully specific RNase T1 piece in the Pfizer-BioNTech vaccine (Comirnaty).
• Isomeric digestion pieces. There are also isomers of ACAAG. AACAG appears three times and CAAAG appears once in Comirnaty.
• Similar MS2 spectra. The MS2 spectra of ACAAG and AACAG differ in only a few ions, those that fragment between the 2nd and 3rd nucleotides. And even some of these ions are ambiguous, as the ions for AC in ACAAG may also appear as internal fragments in the MS2 spectrum of AACAG.

Digestion-specific pieces of unique mass, different from any other digestionspecific piece, are especially advantageous, because they can be identified by MS1 alone. But how frequent are unique-mass pieces in a long mRNA? How does their frequency vary with mRNA length, piece length, and digestion efficiency? How many MS2 peaks are needed to distinguish isomers?