Systems vaccinology of the BNT162b2 mRNA vaccine in humans P ra bh u S. A ru na ch al am , M ad el ei ne K. D.

A delayed ion extraction technique is shown to dramatically improve mass resolution and the overall quality of matrix-assisted laser desorption ionization (MALDI) mass spectra of oligonucleotides. Isotope limited mass resolution was obtained on samples up to 10-kDa molecular mass in linear mode, and as high as 7500 mass resolution (defined at half peak height) was observed in reflector mode. This performance is as good as that achieved to date for peptides and proteins. Applications included the detection of oxidized byproducts of phosphorothioate DNA and separation of components differing only by 15 Da at 9.5-kDa molecular mass. In addition to single components, complex mixtures could also be analyzed at greatly improved performance over conventional MALDI. An example is shown for sequence verification of an oligonucleotide of 31 bases in length by analyzing the failure products. Mass accuracy was adequate to verify sequences of oligodeoxyribonucleotides up to 9500-Da molecular mass. Fast fragmentation taking place between the ionizing pulse and the extraction pulse is demonstrated to be a sequencing tool for small oligonucleotides. By proper selection of matrix material, wavelength, and irradiance, fast fragmentation can be promoted efficiently. Fragment ions tend to form from cleavage of phosphodiester bonds, as previously observed in infrared MALDI.

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Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40-and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has the potential to rapidly acquire DNA sequence information (1-3). MALDI-MS has already been successfully applied to confirmation of the sequence of short synthetic oligonucleotides by mass analysis of their associated synthesis failure products (4) and by mass analysis of fragments generated in a time-dependent exonuclease digestion (5). With these methods, identification of the sequence depends on determination of the mass of each fragment with sufficient accuracy to identify each base by its unique mass. These methods have been applied only to relatively short oligonucleotides, because the resolution and sensitivity of MALDI-MS generally falls off dramatically with increasing size.In contrast, sequence determination from separate Sanger dideoxy termination reactions only requires resolution sufficient to sequentially distinguish termination fragments of length n from length n + 1. Using this method, mass spectra of each of the four specific dideoxy termination reactions generated from Sanger chemistry are overlaid and each sized product correlated to one of the four base termination reactions.