Proteomics is essential for deciphering how biomolecules interact as a system and for understanding the functions of cellular systems in human diseases. However, the unique characteristics of the human proteome, which include the large dynamic range of protein expression and the extreme complexity resulting from a plethora of post-translational modifications (PTMs) and sequence variations, make such analyses challenging. A comprehensive analysis of all proteoforms in the human proteome that arise from genetic variations, alternative splicing, and PTMs, is essential for gaining a transformative understanding of disease mechanisms and identifying new therapeutic targets. The emerging top-down mass spectrometry (MS)-based proteomics, which is based on analysis of intact proteins, is arguably the most powerful method to comprehensively characterize proteoforms to decipher the PTM codes together with genetic variations. However, top-down MS-based proteomics still faces significant challenges in terms of protein solubility, protein separation, and detection of low- abundance proteins. In this multiple-PI project, we will develop novel approaches enabled by nanotechnology and materials chemistry to overcome the challenges facing top-down proteomics in a comprehensive manner. The specific objectives of this proposal are: 1) To develop novel top-down MS-compatible surfactants that can effectively solubilize all categories of proteins including membrane proteins. 2) To develop novel chromatography materials based on mesoporous silica nanomaterials with uniform pore sizes and new strategies for high- resolution multi-dimensional liquid chromatography for effective separation of intact proteins. 3). To develop functionalized multivalent nanoparticles for highly specific enrichment of low-abundance proteins and proteins with PTMs. Our highly interdisciplinary approach is a perfect marriage between materials chemistry/nanotechnology and top-down MS-based proteomics. It is built on an existing collaboration between the two PIs that has led to significant preliminary results. We envision the development of the proposed technologies will significantly advance the burgeoning field of top-down proteomics. The success in our research will provide innovative tools for gaining transformative insights into the molecular basis of diseases and developing new strategies for the early diagnosis, prevention, and better treatment of human diseases.

Public Health Relevance

Proteomics is essential for deciphering how biomolecules interact as a system and for understanding the function of cellular systems in human disease. This proposal seeks to develop novel approaches enabled 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, discover new biomarkers for disease diagnosis, and identify new therapeutic targets for better treatment of diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM117058-02
Application #
9147629
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Sheeley, Douglas
Project Start
2015-09-25
Project End
2019-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
2
Fiscal Year
2016
Total Cost
$295,476
Indirect Cost
$97,976
Name
University of Wisconsin Madison
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Yang, Libang; Gregorich, Zachery R; Cai, Wenxuan et al. (2017) Quantitative Proteomics and Immunohistochemistry Reveal Insights into Cellular and Molecular Processes in the Infarct Border Zone One Month after Myocardial Infarction. J Proteome Res 16:2101-2112
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
Gregorich, Zachery R; Cai, Wenxuan; Lin, Ziqing et al. (2017) Distinct sequences and post-translational modifications in cardiac atrial and ventricular myosin light chains revealed by top-down mass spectrometry. J Mol Cell Cardiol 107:13-21
Wu, Cheng-Guo; Chen, Hui; Guo, Feng et al. (2017) PP2A-B' holoenzyme substrate recognition, regulation and role in cytokinesis. Cell Discov 3:17027
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
Cai, Wenxuan; Tucholski, Trisha; Chen, Bifan et al. (2017) Top-Down Proteomics of Large Proteins up to 223 kDa Enabled by Serial Size Exclusion Chromatography Strategy. Anal Chem 89:5467-5475
Chen, Bifan; Hwang, Leekyoung; Ochowicz, William et al. (2017) Coupling functionalized cobalt ferrite nanoparticle enrichment with online LC/MS/MS for top-down phosphoproteomics. Chem Sci 8:4306-4311
Hu, Chia-Wei; Worth, Matthew; Fan, Dacheng et al. (2017) Electrophilic probes for deciphering substrate recognition by O-GlcNAc transferase. Nat Chem Biol 13:1267-1273
Jin, Yutong; Peng, Ying; Lin, Ziqing et al. (2016) Comprehensive analysis of tropomyosin isoforms in skeletal muscles by top-down proteomics. J Muscle Res Cell Motil 37:41-52
Cai, Wenxuan; Tucholski, Trisha M; Gregorich, Zachery R et al. (2016) Top-down Proteomics: Technology Advancements and Applications to Heart Diseases. Expert Rev Proteomics 13:717-30

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