Practical Mass Spectrometry Upgrade for Identifying Fragile Protein Modifications by ECD

SummaryThe speedresolutionand mass accuracy of modern mass spectrometers have revolutionized proteomicsbut the accurate identification and quantification of post translational modificationsPTMsremain a major challenge that ultimately limits many current biomedical and pharmaceutical applicationsA pivotal weakness lies in the almost exclusive use of collision induced dissociationCIDto induce fragmentation because most PTMssuch as phosphorylationhave labile bonds that are commonly lost in complex ways when subjected to CIDFurthermoreCID limits proteomics to bottom up analyses of trypsindigested peptides ofresiduesIt is well established that an alternative fragmentation methodology called electron capture dissociationECDcan produce exceptionally clean spectra that preserve PTMsbut this technique is currently feasible only in expensive FTICR mass spectrometersProviding enough lowenergy electrons to efficiently fragment peptides hasuntil nowfundamentally limited the application of ECDWe have developed an ECD cell that operates without affecting the ion flight path of conventional mass spectrometersBased on that new technologyour Phase I SBIR project was designed to at least double fragmentation efficiency by exploiting the distinctive geometry of Orbitrap mass spectrometers to enable ions to make two passes through the ECD cellWe exceeded our Phase I milestones by demonstrating that our ECD cell quadrupled efficiencydue in part to ions moving slower through our cell in the Orbitrap than in other types of mass spectrometersWe further showed that our ECD cell was easily installed in Orbitraps in an hour without affecting the instrumentsandaposperformanceWe established the ECD works particularly well for the analysis of native proteinseven for top down hydrogen deuterium structural analysesFor Phase IIourst aim is to refine each of the elements in the ECD cell to integrate easily in four Orbitrap family members and then to exploit the cellandapos s capabilities to produce high energy electrons to achieve stronger fragmentation by Electron Induced DissociationEIDOurnd aim involves working with early adopters to develop the technology for commercial release and validate its substantial advantages over competing technologiesAdoption of our technology will accelerate the ability of many NIH investigators to probe disease mechanisms and identify diagnostic therapeutic biomarkers with increased speed and accuracy that will result in fewer mistaken identifications in complex biological samplesOur immediate commercial objective for Phase III is to provide cost effective upgrade kits for theOrbitraps in serviceThe longerrange commercial goal is to develop fully integrated solutions that will enable the biopharmaceutical industry to characterize therapeutic protein products such as antibody conjugated drugsand to validatebiosimilarsfor the FDA and other regulatory agencies Project NarrativeEven with all of the scientific progress we have made to datethe complexity of disease affected tissues still challenges our ability to probe what makes people illThe goal of this Phase II SBIR project is to extend the Phase I progress toward developing and commercializing a powerful new tool for more effectively cutting large biological molecules into identifiable piecesPhase II success will allow us to engagePhase IIIcommercialization partners and customers with a next generation technology that will improve the diagnosis and treatment of diseases ranging from arthritiscancer and diabetes to heart disease and neurodegeneration