Inert Gas Modulation of Electron-Based Fragmentation for Proteomics

The identification and quantification of biological macromolecules remain challenging despite major advances in the speedresolution and mass accuracy of modern mass spectrometersA key weakness with current instrumentation lies in the methods used to induce fragmentationThe reliance in particular on collision induced dissociationCIDhas limited such analyses to bottom up workflows of trypsin digested peptides ofresiduesWhen subjected to CIDmany fragile PTMs on these short peptides are lost in complex fragmentation channelsAn alternative fragmentation methodology called electron capture dissociationECDis well known for producing exceptionally clean spectra of entire proteins while also preserving PTMsThe difficulty arises from confining enough low energy electrons to efficiently fragment peptide bondswhich has prevented its adoption in most mass spectrometersAt e MSionwe have developed an efficient electron fragmentation technology called ExD to confine electrons using only DC static fields and a carefully sculpted magnetic fieldTwo major advantages of our technology over competing fragmentation techniques such as ETD are speed and simplicity and we are achieving remarkable results with large native proteinsHoweverthe remaining challenge for the widespread adoption of our technology is the relatively lower efficiency with doubly and triply charged peptideswhich remain the core activity for most proteomic facilitiesWhile testing our ExD cell attached directly to the high gas pressure ion mobility cell in the Waters Synapt Gwe have discovered that that having nitrogen gas flow through our cell can increase fragmentation efficiency for doubly charged peptides fromto overThe feasibility question we pose for this phase I application is how to best use gas flow to maximize the fragmentation of peptides and other low charged molecules in our ExD cellTo accomplish this objectiveour primary aim is to optimize the ExD cell design for introduce gas near the filament chamber to better thermalizecoolelectrons to improve electron captureWe hypothesize inert gas near the filament can better distribute electrons within the cell to improve electron captureResults from these aims will establish how to dramatically improve electron based fragmentation for bottom up proteomics in mass spectrometryThe adoption of our technology is an extremely cost effective solution that will accelerate the ability of many NIH investigators to probe disease mechanismsto characterize complex macromolecules in biological samples with increased accuracy and speedreduce the rate of false discoveries and misidentificationsand reveal new details not possible by current approaches Even with all of the scientific progress made to datethe complexity of disease affected tissues still challenges our ability to probe what makes people sickThe goal of this Phase I SBIR project is to develop a powerful tool for more effectively cutting biological molecules into identifiable fragments that will improve the diagnosis and treatment of diseases ranging from arthritiscancer and diabetes to heart disease and neurodegeneration