SBIR-STTR Award

Advances in genome mining, and consequent realization of untapped microbial diversity has lead to renewed interest in natural product (NP) discovery. NPs are a rich source of therapeutic candidates, with a long history of successes in combating infectious diseases, and oncology. The emergence of antibiotic-resistant bacteria like C. difficile and carbapenem-resistant enterobacteiaceae (CRE) only emphasizes the desperate, and growing need for new therapeutics. Investigation of microenvironments for novel NP compounds is labor-intensive, and requires numerous molecular biology, and biochemical techniques to isolate and characterize novel NP compounds. In addition, months of effort to isolate and identify a compound can be wasted if the compound was previously discovered. High-throughput methods are needed to quickly identify drug leads from a sample, with minimal upfront investment. Tandem mass spectrometry (MS) is a high-throughput technology capable of sensitively identifying molecules. Unlike nuclear magnetic resonance spectroscopy, which is a widely used in NP research for structure elucidation and dereplication, tandem MS can be used to analyze small amounts of compounds directly from the environmental extract. While MS offers the hope of rapid NP identification, 98% of spectra cannot be matched to existing spectral libraries. Quorum is a computational platform that organizes tandem MS data into molecular networks. Molecular networks connect related compounds that differ by a mutation, modification, or adduct. Quorum aims to increase NP compound annotations compared to previous approaches by integrating spectral library search and structure database search. In addition, annotations of known compounds can be automatically propagated to related, but unknown compounds via similarity of their tandem mass spectra. Quorum will be deployed as a web application to enable interactive visual interrogation of the network topology and underlying data analysis. Given the design of a MS experiment (e.g. case versus control or time-series), the application will perform statistical analysis to highlight significantly enriched compounds and sub-networks. These features can be used to quickly identify novel drug leads or to optimize growth and extraction protocols.

Public Health Relevance Statement:
While natural products and their derivatives comprise 45% of FDA recently approved drugs, new discoveries by pharmaceutical companies has waned due to the difficulty in isolating novel compounds and lack of high-throughput methods for characterization. Quorum is a software tool that analyzes mass spectrometry data to characterize compounds from microbe rich extracts, as well as track compound behavior across samples or conditions. The innovative molecular networking approach employed by Quorum will dramatically increase the rate for identifying compounds and accelerate discovery of new therapeutics.

Project Terms:
adduct; Anti Inflammatory Analgesics; Anti-Bacterial Agents; Antibiotic Resistance; bacterial resistance; Behavior; Biochemical; Biological; carbapenem resistance; Catalogs; Chemical Structure; Clostridium difficile; combat; Communicable Diseases; Complex; Data; Data Analyses; database structure; Databases; design; Development; digital; Dolastatin Compound; drug discovery; drug resistant bacteria; Environment; environmental stressor; experimental study; Family; follow-up; Genome; Growth; high throughput technology; Imagery; improved; innovation; interest; Investigation; Investments; Lead; Libraries; Lipids; Malignant Neoplasms; Manuals; Mass Spectrum Analysis; member; Methods; Microbe; microbial; Mining; Modification; Molecular; Molecular Biology; Multivariate Analysis; Mutation; Natural Increases; Natural Product Drug; Natural Products; NMR Spectroscopy; novel; novel therapeutics; oncology; Pattern; Peptides; Pharmaceutical Preparations; Pharmacologic Substance; programs; Proteomics; Protocols documentation; Recording of previous events; Research; Research Personnel; Resources; Sampling; Series; social; Software Tools; Source; Statistical Data Interpretation; Structure; success; tandem mass spectrometry; Techniques; Technology; Therapeutic; therapeutic candidate; Time; tool; Visual; web app

Glycosylation is a pervasive modification estimated to modify more than 50% of human proteins and is a key component of host-pathogen interactions, adaptive and innate immunity, and therapeutic development. Computational tools for glycoprotein analysis make simplifying assumptions about possible glycan structures, limiting their utility in many scenarios. This proposal aims to comprehensively characterize glycoproteins, including novel and unexpected glycans, with Digital Proteomics’ Quorum mass spectrometry data analysis software, and enable further glycoprotein characterization through the development of antibodies targeting discovered glycopeptides. The Quorum software builds on the spectral networking paradigm that posits that structurally similar molecules produce similar mass spectra. In Quorum for glycopeptide analysis, networked spectra represent different glycan structures on the same glycosite of a protein. Similar to traditional approaches to glycopeptide identification, Quorum matches glycopeptide spectra to a protein sequence database and glycan structure database. Unlike traditional approaches, Quorum employs a rigorous false-discovery rate calculation based on variant glycan structures in target-decoy analysis. In addition, Quorum increases the identification rate of glycopeptides by propagating identifications along edges in the network, enabling the discovery of glycan structures that are not in the structure database and may be completely novel. Quorum will be benchmarked on glycoprotein standards, well-characterized NIST monoclonal antibody reference material, and total IgG which will establish a baseline for spectral network variant glycopeptide discovery. The Quorum software is deployed to the cloud, as part of our extensible platform Quorum Cloud, where the glycoprotein analysis is a plugin to generic spectral networking analysis. Enlisted testers of the cloud platform have applications in glycoengineering, glycoenrichment assays, and therapeutic development.

Public Health Relevance Statement:
Glycans are key modulators of protein stability and function, and play critical roles in host-pathogen interactions, cancer metastasis, and immunology. Research into protein glycosylation is hampered by inadequate tools to identify the full diversity of glycan structures on a proteins. We are developing Quorum, a software tool to accurately characterize glycan structures on proteins to provide important information for therapeutic and diagnostic development.

Project Terms:
adaptive immunity; Algorithms; Amino Acid Sequence Databases; analytical tool; Antibodies; Antigens; assay development; base; Basic Science; Benchmarking; CA-125 Antigen; cancer biomarkers; Characteristics; Cleaved cell; Cloud Computing; cloud platform; cluster computing; Collection; Communities; Complex; Computer software; computerized tools; Data; Data Analyses; Data Set; database structure; Databases; Development; Diagnostic; digital; Discipline; Discrimination; Disease; Disease Progression; Drug Kinetics; Engineering; Glycopeptides; Glycoproteins; glycoproteomics; glycosylation; Half-Life; Health; Human; Immunoglobulin G; Immunology; improved; instrumentation; interest; Ions; Knowledge; Libraries; Link; macromolecule; malignant breast neoplasm; Malignant neoplasm of ovary; Mass Spectrum Analysis; materials science; Metadata; Methods; Modification; Monitor; Monoclonal Antibodies; Monosaccharides; Natural Immunity; Natural Products; Neoplasm Metastasis; Noise; novel; pathogen; Pathway Analysis; peptide I; Peptides; Performance; Pharmacologic Substance; Phase; Play; Polysaccharides; Protein Analysis; Protein Glycosylation; protein phosphatase inhibitor-2; Proteins; Proteome; Proteomics; prototype; Publications; Reporting; Research; Research Personnel; Role; Signal Transduction; Site; Small Business Innovation Research Grant; Software Tools; Structure; tandem mass spectrometry; Technology; Therapeutic; therapeutic development; therapeutic protein; tool; translational impact; tumor; Urine; Variant