Sequencing of DNA Mixtures Using Liquid Chromatography─Postactivated Plasma Electron Detachment Dissociation Mass Spectrometry

We previously reported fast electron detachment dissociation (EDD) of oligonucleotides in electron-cation neutral plasma (plasma EDD or pEDD), which showed potential for sequencing oligonucleotides with high coverage [ <i>Anal. Chem.</i> 2022, 94, 15510-15517]; however, the fragment intensities were weak because charge-reduced species (CRS_<i>n</i>) were the dominant products, resulting in low sensitivity. When we applied LC-pEDD MS/MS to DNA mixtures in a data-dependent acquisition (DDA) workflow, the total sequence coverage was lower than that of conventional collision-induced dissociation (CID). In this work, we enhanced the sensitivity of pEDD by applying collisional postactivation (PA) to first CRS: [M - <i>n</i>H]^{•(<i>n</i>-1)-} (CRS₁) and/or second CRS: [M - <i>n</i>H]^{(<i>n</i>-2)-} (CRS₂). We initially expected a radical CRS₁ to have a significantly lower dissociation energy threshold than the nonradical precursors, [M - <i>n</i>H]^<i>n</i>-, analogous to electron capture dissociation (ECD) on protonated peptides and proteins; however, the threshold of CRS₁ was substantially identical to that of the precursors. We also optimized the PA energy for CRS₁ with various charge states. We applied the optimized postactivated plasma-EDD (PA-pEDD) to DNA mixtures in a DDA workflow. The LC-DDA-MS/MS workflow, with the optimized PA-pEDD, enhanced the sensitivity of fragment intensities, resulting in 100% sequence coverage for many DNAs in the mixtures.