Efficient, Zero Scrambling Fragmentation of Deuterium Labeled Peptides on the ZenoToF 7600 Electron Activated Dissociation Platform

Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) has become an increasingly important tool in protein research, with large-scale applications in biopharmaceutical development and manufacturing. One of the limitations of classical bottom-up HDX is that it usually provides a "peptide-averaged" picture of structure and dynamics, rather than site-specific (i.e., individual amino acid-level) information. A major challenge for site-specific HDX-MS analyses has been that classical fragmentation techniques such as CAD invariably cause random redistribution of the deuterium labels across the peptide backbone, known as deuterium scrambling. Several groups have demonstrated that this problem can be overcome using nonergodic fragmentation and "cool" ion flight conditions. A major hurdle to widespread adoption of this approach, however, is that the exceedingly low fragmentation efficiency of electron capture dissociation (ECD) combined with the lower transmission efficiency of "cool" ion flight conditions impose a very strong attenuation on sensitivity, to the point where this method becomes impractical for many "real-world" applications. Here, we introduce a workflow and instrument conditions on the Sciex 7600 ZenoToF electron activated dissociation (EAD) platform that allow for zero scrambling ECD fragmentation with limited (and in some cases no) sensitivity loss. We expect that this workflow will be ideal for broadly applicable, site-specific HDX-MS analyses using a middle-down workflow.