Activation of Oligonucleotide Polyanions Using Collisions, Electrons and Photons in a timsOmni Platform

Abstract

We describe here various ion activation experiments realized in the Omnitrap platform integrated on the timsOmni mass spectrometer for the analysis of oligonucleotides in the negative ion mode. The activation methods include resonance collision-induced dissociation (RCID), electron detachment dissociation (EDD), infrared laser multiple-photon activation (IRMPD) and UV laser photodissociation (UVPD) at 266 nm. Special emphasis is given to EDD, either as a standalone technique or in conjunction with vibrational reactivation of the ion radicals. We describe EDD on standard 6-mer DNA sequences that have been extensively characterized on other instruments, followed by a comparison of several activation approaches for the phosphorothioate-based oligonucleotide therapeutics Fomivirsen, and conclude with the fragmentation analysis of 46-mer DNA and RNA. EDD alone already provides excellent sequence information on short oligonucleotides such as Fomivirsen, but MS3 combinations such as EDD-RCID or EDD-IRMPD proved even more effective, including for the 46-mer DNA (less prone to fragmentation than RNA) at a relatively low charge states. The diversity of ion activation combinations available on the Omnitrap platform is demonstrated by MS4 experiments, which experimentally validate the formation of d ions from a• radical fragments, and w ions from z• radical fragments produced by EDD.