on 04-06-201704:29 PM - edited on 10-15-202111:32 AM by AnalyteGuru
Nicholas M. Riley,1,2 Michael S. Westphall,1 Alexander S. Hebert,1 and Joshua J. Coon1-4,* Anal. Chem., Just Accepted Manuscript Using concurrent IR photo-activation during electron transfer dissociation (ETD) reactions, i.e., activated ion ETD (AI-ETD), significantly increases dissociation efficiency resulting in improved overall performance. Here we describe the first implementation of AI-ETD on a quadrupole-Orbitrap-quadrupole linear ion trap (QLT) hybrid MS system (Orbitrap Fusion Lumos) and demonstrate the substantial benefits it offers for peptide characterization. First, we show that AI-ETD can be implemented in a straight-forward manner by fastening the laser and guiding optics to the instrument chassis itself, making alignment with the trapping volume of the QLT simple and robust. We then characterize the performance of AI-ETD using standard peptides in addition to a complex mixtures of tryptic peptides using LC-MS/MS, showing not only that AI-ETD can nearly double the identifications achieved with ETD alone, but also that it outperforms the other available supplemental activation methods (ETcaD and EThcD). Finally, we introduce a new activation scheme called AI-ETD+ that combines AI-ETD in the high pressure cell of the QLT with a short infrared multi-photon dissociation (IRMPD) activation in the low pressure cell. This reaction scheme introduces no addition time to the scan duty cycle but generates MS/MS spectra rich in b/y-type and c/z●-type product ions. The extensive generation of fragment ions in AI-ETD+ substantially increases peptide sequence coverage while also improving peptide identifications over all other ETD methods, making it a valuable new tool for hybrid fragmentation approaches.