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 #
5R01GM117058-03
Application #
9336949
Study Section
Instrumentation and Systems Development Study Section (ISD)
Program Officer
Smith, Ward
Project Start
2015-09-25
Project End
2019-08-31
Budget Start
2017-09-01
Budget End
2018-08-31
Support Year
3
Fiscal Year
2017
Total Cost
Indirect Cost
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
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
Wu, Zhijie; Tiambeng, Timothy N; Cai, Wenxuan et al. (2018) Impact of Phosphorylation on the Mass Spectrometry Quantification of Intact Phosphoproteins. Anal Chem 90:4935-4939
Chen, Zhilong; Song, Jiangping; Chen, Liang et al. (2018) Characterization of TTN Novex Splicing Variants across Species and the Role of RBM20 in Novex-Specific Exon Splicing. Genes (Basel) 9:
Chen, Bifan; Lin, Ziqing; Alpert, Andrew J et al. (2018) Online Hydrophobic Interaction Chromatography-Mass Spectrometry for the Analysis of Intact Monoclonal Antibodies. Anal Chem 90:7135-7138
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
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
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
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

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