Practical Mass Spectrometry Upgrade for Identifying Fragile Protein Modifications by ECD

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The speed, resolution and high mass accuracy of modern mass spectrometers have revolutionized proteomics, particularly for determining fragile post-translational modifications that control most cellular processes. Accurate identification and quantitation of phosphorylation sites remain a major challenge in proteomics. The key weakness with mass spectrometry for phospho-proteomics lies in the methods used to induce fragmentation, because phosphoryl bonds are among the most labile chemical bonds in proteins and are lost in complex ways by current collision-based fragmentation approaches. An alternative fragmentation methodology called electron capture dissociation (ECD) is well established to produce exceptional spectra of phosphopeptides, but is currently feasible only in expensive FTICR mass spectrometers. The fundamental limitation to ECD is providing enough low-energy electrons to efficiently fragment peptides. We have two issued patents protecting a new technology enabling a practical ECD cell that uses carefully sculpted magnetic fields to confine electrons. The ECD cell is only two centimeters in length and can be readily incorporated into virtually any mass spectrometer. The major factor limiting adaption with our ECD cell is that the efficiency is limited to 5-10% for doubly charged phosphopeptides. This raise concerns about the loss of sensitivity for low abundance peptides. However, peptides now fly just once through the cell. Our Phase-I proposes to increase this efficiency by reflecting ions to make multiple passages through the ECD cell. For this purpose, we will focus on Orbitrap mass spectrometers, which have become the most widely used instruments for proteomics. Their unique design allows integration of the ECD cell without changing any component in the Orbitrap itself. The feasibility question to be answered in Phase-I is: how to best incorporate the ECD cell to pass peptides and proteins through the cell multiple times to increase fragmentation efficiency? The challenge is to avoid losing sensitivity because of peptide ions scattering as they are reflected. In Phase-II, we will work with early- adopters to validate the cell for quantifying post-translational modifications by both top-down and bottom-up proteomic approaches. Supporting letters are included from the discoverer of ECD, the inventor of the Orbitrap, and two internationally known leaders of proteomics. Phase-III will be to provide cost-effective upgrade kits for the thousands of Orbitraps currently in operation. As the technology wins acceptance, our company will develop new generations of mass spectrometers capitalizing on the additional information provided by ECD. The adoption of our technology for a modest cost will accelerate the ability of many NIH investigators to probe disease mechanisms as well as identify diagnostic and therapeutic biomarkers with increased accuracy, greater speed and fewer mistakes.