In the post-genomics era, a comprehensive analysis of ?proteoforms? that arise from genetic variations and post-translational modifications (PTMs) is essential for understanding biological systems at a functional level and for dissecting complex molecular systems with consideration of individual variability for precision medicine. Top- down mass spectrometry (MS)-based proteomics that analyzes intact proteins is the most powerful method to comprehensively characterize proteoforms to decipher the PTM codes together with sequence variations. Although significant strides have been made recently in both MS hardware and software to advance top-down MS closer to the mainstream, top-down proteomics still faces major challenges. In particular, the proteome is extremely complex and has a high dynamic range in addition to the low solubility of many proteins, making it highly challenging for high-throughput proteomic study. Building on the success in the last funding period, in this multiple-PI renewal application, we will continue to develop innovative strategies empowered by nanotechnology and materials/organic chemistry to further address the challenges in top-down proteomics. The specific objectives of this proposal are: 1) To address the protein solubility challenge, we will develop a novel strategy enabled by a photocleavable surfactant for extracellular matrix (ECM) proteomics; and design, synthesize, and evaluate a novel class of photocleavable nonionic surfactants that can retain the native structures of proteins for native MS-based top-down proteomics. 2) To address the high dynamic range challenge, we will develop novel surface functionalized magnetic nanoparticles (NPs) to mimic antibodies for capturing and enriching low abundance proteins, such as cardiac troponin I (cTnI, a gold-standard biomarker for heart diseases) from tissues/blood and G-protein coupled receptors (GPCRs, a major class of drug targets) from cells/tissues, for downstream comprehensive analysis of all proteoforms by top-down proteomics. Our highly interdisciplinary approach integrates materials chemistry/nanotechnology with top-down MS-based proteomics, and is based on an existing productive collaboration between two PIs that has led to significant progress and publications from the past funding period. Success in our proposed research will provide innovative tools to enable top-down proteomics of poorly soluble and low abundance proteins, which will lay important technological foundation for understanding the critical role that ECM plays in disease progression in cancer and cardiac diseases, defining the structure-function relationship of native membrane complexes, developing a comprehensive cTnI assay for the diagnosis of cardiac diseases with high accuracy, and understanding the important roles of GPCR signaling during the onset of numerous human diseases including cancer, diabetes, and cardiovascular diseases.

Public Health Relevance

Proteomics is essential for dissecting complex molecular systems with consideration of individual variability for precision medicine. This proposal seeks to develop novel approaches empowered by nanotechnology and materials chemistry to overcome the challenges facing top-down proteomics. Success in the proposed research will provide innovative tools to gain transformative insights in molecular basis of diseases, identify new therapeutic targets for better treatment of diseases, and provide comprehensive diagnostic assays for cardiac diseases to enable precision medicine.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
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Gindhart, Joseph G
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University of Wisconsin Madison
Schools of Arts and Sciences
United States
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Chen, Bifan; Brown, Kyle A; Lin, Ziqing et al. (2018) Top-Down Proteomics: Ready for Prime Time? Anal Chem 90:110-127
Cai, Wenxuan; Hite, Zachary L; Lyu, Beini et al. (2018) Temperature-sensitive sarcomeric protein post-translational modifications revealed by top-down proteomics. J Mol Cell Cardiol 122:11-22
Lin, Ziqing; Guo, Fang; Gregorich, Zachery R et al. (2018) Comprehensive Characterization of Swine Cardiac Troponin T Proteoforms by Top-Down Mass Spectrometry. J Am Soc Mass Spectrom 29:1284-1294
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Jin, Yutong; Wei, Liming; Cai, Wenxuan et al. (2017) Complete Characterization of Cardiac Myosin Heavy Chain (223 kDa) Enabled by Size-Exclusion Chromatography and Middle-Down Mass Spectrometry. Anal Chem 89:4922-4930
Wu, Cheng-Guo; Chen, Hui; Guo, Feng et al. (2017) PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis. Cell Discov 3:17027
Rana, Ambar S J B; Ge, Ying; Strieter, Eric R (2017) Ubiquitin Chain Enrichment Middle-Down Mass Spectrometry (UbiChEM-MS) Reveals Cell-Cycle Dependent Formation of Lys11/Lys48 Branched Ubiquitin Chains. J Proteome Res 16:3363-3369
Chen, Bifan; Guo, Xiao; Tucholski, Trisha et al. (2017) The Impact of Phosphorylation on Electron Capture Dissociation of Proteins: A Top-Down Perspective. J Am Soc Mass Spectrom 28:1805-1814

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