SBIR-STTR Award

The speed, resolution and high mass accuracy of modern mass spectrometers have revolutionized proteomics, but the accurate identification and quantitation of post-translational modifications (PTMs) remain a major challenge—a key limitation for many important medical applications. A key weakness with current mass spectrometry for proteomics lies in the methods used to induce fragmentation, because PTMs such as phosphorylation 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 exceptionally clean spectra that preserve PTMs, but is currently feasible only in expensive FTICR mass spectrometers. The fundamental limitation to ECD is the difficulty of providing enough low-energy electrons to efficiently fragment peptides. We have discovered how to use carefully sculpted magnetic fields with a hot electron-producing filament to restrain large numbers of electrons in the flight path of ions. This can be adapted in any common tandem mass spectrometer without changing the existing ion optics, but our best designs can only fragment 3-5% of doubly charged trypsin-digested peptides—the most common workflow used in mass spectrometry. This low fragmentation efficiency limits sensitivity, which has proved to be the major barrier to adopting this powerful methodology by the mass spectrometry industry. The key focus of this Phase I SBIR project is determining how to increase the interaction time of ions with electrons confined to a narrow beam by the magnetic fields to prove this concept feasible. The reaction time currently is 1-2 microseconds. Our Phase I feasibility question is whether fragmentation can be effectively increased at least two-fold by transiently stopping peptide ions in the ECD cell without significant loss due to electrostatic scattering. In addition, the design must retain the sub-millisecond speed necessary to be compatible for current front-end HPLC and ion mobility separations used with mass spectrometers for complex samples. Rigorous computer simulations show these objectives can be accomplished by carefully cooling precursor ions and then transiently stopping their flight with carefully timed electrical pulses to electrostatic lenses. Proof of feasibility and validated concept demonstration (Phase II) are essential in engaging the major instrument manufacturers to further develop and commercialize our ECD technology for use in their mass spectrometer products. Success will also show how our technology can produce better fragmentation of the most challenging analytes analyzed by mass spectrometry, including lipids, glycans, and other difficult-to- fragment drugs/metabolites. The adoption of our technology will accelerate the ability of many NIH investigators to probe disease mechanisms and identify diagnostic/therapeutic biomarkers with increased accuracy and greater speed, while making fewer mistaken identifications in complex biological samples.

Public Health Relevance Statement:


Project narrative:
The enormous complexity of disease-affected tissues presents major problems for figuring out what is wrong. The goal of this research is to provide a powerful molecular tool to more effectively cut biological molecules into identifiable pieces. The ultimate goal of this multi-phase SBIR project is to develop, validate, and commercialize next-generation tools that will facilitate key advances in disease diagnosis and treatment.

Project Terms:
Acetylation; Address; Adopted; Adoption; Affect; Amides; base; Biological; Carbohydrates; Cardiovascular Diseases; Cells; Charge; chemical bond; Cleaved cell; Complex; Computer Simulation; density; design; Detection; Deuterium; Diagnostic; Disease; disease diagnosis; Dissociation; electron energy; Electrons; Electrostatics; experimental study; Filament; Fourier transform ion cyclotron resonance; Goals; High Pressure Liquid Chromatography; Hydrogen; improved; Industry; Inflammation; instrument; ion mobility; Ions; Isotope Labeling; Isotopes; lens; Lipids; magnetic field; Malignant Neoplasms; Manufacturer Name; mass spectrometer; Mass Spectrum Analysis; Measures; Medical; Methodology; Methods; millisecond; Modernization; Modification; Molecular; Movement; Nerve Degeneration; next generation; Optics; Pattern; Peptide Fragments; Peptides; Pharmaceutical Preparations; Phase; Phosphopeptides; Phosphorylation; Physiologic pulse; Polysaccharides; Post-Translational Protein Processing; Power Sources; Process; programs; Promega; Proteins; Proteomics; Radiation; Reaction Time; Research; research and development; Research Personnel; Resolution; Sampling; Small Business Innovation Research Grant; Speed; success; tandem mass spectrometry; Technology; therapeutic biomarker; Therapeutic Intervention; Time; Tissues; tool; transmission process; TRAP Peptide; Trypsin; United States National Institutes of Health; Work

The primary market focus for the high-end mass spectrometer industry is to fully characterize proteins and their post-translational modifications (PTMs) within the biopharmaceutical industry. These analyses remain challenging despite major advances in the speed, resolution and mass accuracy of modern mass spectrometers. A key weakness with current instrumentation for protein characterization lies in the methods used to induce fragmentation. The reliance in particular on collision-induced dissociation (CID) has limited such analyses to bottom-up workflows of trypsin-digested peptides of 10-30 residues. When subjected to CID, many fragile PTMs on these short peptides are lost in complex ways. An alternative fragmentation methodology called electron capture dissociation (ECD) is well known for producing exceptionally clean spectra of entire proteins while also preserving PTMs. However, this technology has been feasible only in expensive FTICR mass spectrometers. The difficulty arises from confining enough low-energy electrons to efficiently fragment peptide bonds, which has limited the application of ECD in other instruments. The e-MSion team has developed an efficient ECD technology to confine electrons with a carefully designed magnetic field that operates without affecting the ion flightpath in mass spectrometers. One major advantage of our technology over competing fragmentation techniques such as ETD is speed. We established Phase I feasibility by showing that our ECD technology is fast enough to be used in quadrupole-Time of Flight (Q- ToF) mass spectrometers at speeds compatible with UPLC and ion mobility-based separations of complex samples. Our technology also efficiently supports sequencing of proteins as large as 30 kDa in seconds while leaving even the most fragile PTMs intact. The proposed Phase II SBIR project will complete the optimization/integration of our ECD into Q-ToF's to make the operation seamless for two major manufacturers of Q-ToFs. The primary commercial goal is to become a value-added reseller for upgrading Q- Tofs in Phase III. To accomplish this, our first Aim is to refine the engineering, software integration and application to middle- and top-down protein characterization. Aim 2 is to work with early adopters in both Biopharma and in proteomics fields to demonstrate the capabilities of the technology. The third Aim is to further modify the design of the ECD cell to perform Electron-Induced Dissociation (EID) more efficiently for the characterization of singly charged peptides and glycoproteins. This entails subtle modifications to the current ECD cell that allows larger quantities of higher-energy electrons to flow through the system. Completion of Aim 3 will open the market for triple-quad mass spectrometers, which is five times larger than the more expensive Q-ToFs. The adoption of our technology will accelerate the ability of many NIH investigators as well as BioPharma to probe disease mechanisms by characterizing macromolecules in complex biological samples with increased accuracy and speed, while reducing false discoveries.

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 sick. The goal of this Phase II SBIR project is to extend our Phase I progress toward developing and commercializing a powerful 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:
Address; Adoption; Affect; Arthritis; base; Biological; Biological Products; Biological Response Modifier Therapy; Cardiovascular Diseases; Cells; Charge; Chimeric Proteins; Cleaved cell; commercialization; Complex; Computer software; Computers; Consumption; cost; design; Deuterium; Development; Diabetes Mellitus; Diagnosis; Disease; Dissociation; electron energy; Electron Transport; Electrons; Engineering; fly; Fourier transform ion cyclotron resonance; Glycopeptides; Glycoproteins; Goals; Heart Diseases; Hour; Human; Hydrogen; improved; Industry; Inflammation; instrument; instrumentation; Interview; ion mobility; ionization; Ions; Isotopes; Legal patent; macromolecule; magnetic field; Malignant Neoplasms; Manufacturer Name; Marketing; mass spectrometer; Mass Spectrum Analysis; Methodology; Methods; Modernization; Modification; Nerve Degeneration; next generation; off-patent; operation; Pattern; Peptide Fragments; Peptide Sequence Determination; Peptides; Pharmacologic Substance; Phase; phase 1 study; Phosphorylation; Polysaccharides; Post-Translational Protein Processing; preservation; Production; Protein Fragment; Proteins; Proteomics; Quality Control; rapid technique; Reaction; Recombinant Antibody; Research; research and development; Research Personnel; Resolution; Sales; Sampling; Side; Small Business Innovation Research Grant; Speed; success; System; tandem mass spectrometry; Techniques; Technology; Time; Tissues; tool; Trypsin; United States National Institutes of Health; Work