SBIR-STTR Award

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.

Public Health Relevance Statement:
e-MSion, Inc is dedicated to improving the tools used to discovery, better understand, diagnosis and treat disease. Specifically, we will develop a better means of analyzing the most complex biological molecules involved in arthritis, cancer, diabetes, heart disease, and neurodegeneration.

Project Terms:
Address; Adoption; Amides; Arthritis; Back; base; Biological; Businesses; Capital; Cell physiology; Cells; Charge; chemical bond; Cleaved cell; Complex; Computer software; Computers; cost; cost effective; Data; density; design; Diabetes Mellitus; Diagnosis; diagnostic biomarker; Disease; Dissociation; electron energy; Electrons; Engineering; Feedback; fly; Fourier transform ion cyclotron resonance; Generations; Goals; Heart Diseases; improved; Inflammation; Infrared Rays; instrument; International; Ions; Legal patent; Length; lens; Letters; Location; magnetic field; Malignant Neoplasms; Marketing; mass spectrometer; Mass Spectrum Analysis; Measures; metabolomics; Methodology; Methods; Modernization; Modification; Molecular Weight; Nerve Degeneration; new technology; operation; parallelization; Pattern; Peptide Fragments; Peptides; Phase; Phosphopeptides; phosphoproteomics; Phosphorylation; Phosphorylation Site; Post-Translational Protein Processing; Power Sources; prevent; Protein Fragment; Proteins; Proteomics; quantum; Research Personnel; Resolution; Sales; Serine; Small Business Innovation Research Grant; Speed; Technology; Testing; therapeutic biomarker; Threonine; Time; tool; Tyrosine Phosphorylation; Ubiquitin; United States National Institutes of Health; virtual; voltage; Work

The speed, resolution, and mass accuracy of modern mass spectrometers have revolutionized proteomics, but the accurate identification and quantification of post-translational modifications (PTMs) remain a major challenge that ultimately limits many current biomedical and pharmaceutical applications. A pivotal weakness lies in the almost exclusive use of collision-induced dissociation (CID) to induce fragmentation because most PTMs, such as phosphorylation, have labile bonds that are commonly lost in complex ways when subjected to CID. Furthermore, CID limits proteomics to bottom-up analyses of trypsin- digested peptides of 10-40 residues. It is well established that an alternative fragmentation methodology called electron capture dissociation (ECD) can produce exceptionally clean spectra that preserve PTMs, but this technique is currently feasible only in expensive FTICR mass spectrometers. Providing enough low- energy electrons to efficiently fragment peptides has, until now, fundamentally limited the application of ECD. We have developed an ECD cell that operates without affecting the ion-flight path of conventional mass spectrometers. Based on that new technology, our 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 cell. We exceeded our Phase I milestones by demonstrating that our ECD cell quadrupled efficiency, due in part to ions moving slower through our cell in the Orbitrap than in other types of mass spectrometers. We further showed that our ECD cell was easily installed in Orbitraps in an hour without affecting the instruments' performance. We established the ECD works particularly well for the analysis of native proteins, even for top-down hydrogen/deuterium structural analyses. For Phase II, our 1st 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 cell's capabilities to produce high-energy electrons to achieve stronger fragmentation by Electron-Induced Dissociation (EID). Our 2nd aim involves working with early-adopters to develop the technology for commercial release and validate its substantial advantages over competing technologies. Adoption 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 samples. Our immediate commercial objective for Phase-III is to provide cost-effective upgrade kits for the 6,000 Orbitraps in service. The longer- range commercial goal is to develop fully integrated solutions that will enable the biopharmaceutical industry to characterize therapeutic protein products such as antibody-conjugated drugs, and to validate “biosimilars” for the FDA and other regulatory agencies.

Public Health Relevance Statement:


Project narrative:
Even with all of the scientific progress we have made to date, the complexity of disease-affected tissues still challenges our ability to probe what makes people ill. The 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 pieces. Phase II success will allow us to engage “Phase III” commercialization partners and customers with a next-generation technology that will improve the diagnosis and treatment of diseases ranging from arthritis, cancer and diabetes to heart disease and neurodegeneration.

Project Terms:
Acetylation; Address; Adoption; Affect; Antibody-drug conjugates; Arthritis; Australia; base; Biological; Biological Products; Cells; Charge; Chronic Disease; Cleaved cell; commercialization; Complex; Computer software; cost effective; density; design; Detection; Deuterium; Diabetes Mellitus; Diagnosis; Diagnostic; Disease; Dissociation; electron energy; Electrons; Elements; Europe; experience; Family member; Feedback; Foundations; Fourier transform ion cyclotron resonance; Geometry; Goals; Heart Diseases; Hour; Hydrogen; improved; Industry; Inflammation; insight; instrument; Ions; Isotopes; Laboratories; Learning; Letters; Logistics; magnetic field; Malignant Neoplasms; mass spectrometer; Mass Spectrum Analysis; Measures; Methodology; Methods; Mission; Modeling; Modernization; Modification; Nerve Degeneration; new technology; next generation; Pathway interactions; Peptide Fragments; Peptides; Performance; Pharmacologic Substance; Phase; Phosphorylation; Play; Post-Translational Protein Processing; Power Sources; Process; prospective; Proteins; Proteomics; prototype; research and development; Research Personnel; Resolution; Role; Sampling; Services; Shapes; Small Business Innovation Research Grant; Source; Speed; success; Techniques; Technology; Testing; therapeutic biomarker; Therapeutic Intervention; therapeutic protein; Tissues; tool; Travel; Trypsin; United States; United States National Institutes of Health; Work