Implementation of Infrared-Activated Negative Electron Transfer Dissociation (IR-NETD) Using Xenon on a Quadrupole-Orbitrap-Quadrupole Linear Ion Trap Mass Spectrometer.

For tandem mass spectrometry, photoactivation capabilities enable a host of useful dissociation strategies. Herein we present the first implementation of an IR laser system on a next generation, quadrupole-Orbitrap-quadrupole linear ion trap hybrid MS system (Thermo Scientific Orbitrap Ascend). In addition, we establish xenon as an efficient source of radical cations for negative electron transfer dissociation (NETD) reactions. First, we take advantage of the instrument's linear architecture to include a home-built photon detector to improve ease of use and laser alignment, along with straightforward introduction of xenon into the instrument. Second, we assess the instrument performance through infrared-activated NETD (IR-NETD) of a simple, unmodified 6-mer RNA molecule. Finally, we assessed the performance of IR-NETD for characterizing a synthetically complex small interfering RNA molecule, evaluating the effects of parameters including precursor charge state and IR laser power, demonstrating the benefits of concurrent IR photoactivation during NETD reactions. This straightforward instrumental approach represents a powerful and versatile tool for the characterization of complex biopharmaceutical molecules.