The overall goal of our continuing research program is to develop advanced technologies based on electrospray ionization (ESI) with mass spectrometry (MS) to support structural biology efforts. The role of protein assemblies in normal cellular processes and diseases warrants a practical and sensitive method for their study. The structural determination of protein complexes can play an important role in the fundamental understanding of biochemical pathways. New techniques based on native mass spectrometry (i.e., the measurement of biomolecules in their native solution environment to preserve interactions with ligands and other molecules) will be advanced to facilitate the characterization of protein assemblies. Improved methods for measuring large protein complexes (including >0.5 MDa) will be developed. Native protein separations for subsequent analysis by native MS will be advanced. Experimental MS protocols for difficult-to-characterize membrane proteins will be developed. Enhancing multiple charging generated by ESI increases the efficiency for top-down MS (the direct fragmentation of intact gas-phase large molecules to generate structure-informative product ions) and native MS; we will identify new charge enhancing agents, and we will apply these new reagents for top-down MS and membrane protein MS. With high-resolution Fourier transform-ion cyclotron resonance mass spectrometry, enhanced native top-down MS methods will be developed to obtain structurally-relevant information for large protein complexes. Methods incorporating UV photodissociation, electron capture dissociation, and electron ionization dissociation will be developed and tested for their efficiency to generate sequence information from large protein complexes and membrane proteins. Strategies for determining collision cross sections will be improved. These advanced tools will be applied to characterize complexes of biological importance where high-resolution structures are unavailable, including G-coupled protein receptors (GPCRs). Our experimental strategies are broadly applicable to be integrated with different types of biophysical techniques; such integration will allow the study of large and complex molecular machines in greater detail, providing insight into the functional dynamics of the system. Native top-down MS will be a promising approach to advance structural biology and hasten drug discovery and development.

Public Health Relevance

We aim to develop robust analytical technologies that can provide structural information for biologically important proteins, such as proteins involved in neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease). New methodologies to determine binding sites of drugs and other small molecules and proteins to targeted proteins will be developed. The information provided by our methodologies can be used to aid drug development efforts and to better understand biological processes important to human health.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM103479-15
Application #
9769767
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Krepkiy, Dmitriy
Project Start
2004-07-15
Project End
2022-06-30
Budget Start
2019-07-01
Budget End
2020-06-30
Support Year
15
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Nshanian, Michael; Lakshmanan, Rajeswari; Chen, Hao et al. (2018) Enhancing Sensitivity of Liquid Chromatography-Mass Spectrometry of Peptides and Proteins Using Supercharging Agents. Int J Mass Spectrom 427:157-164
Lippens, Jennifer L; Nshanian, Michael; Spahr, Chris et al. (2018) Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes. J Am Soc Mass Spectrom 29:183-193
Wongkongkathep, Piriya; Han, Jong Yoon; Choi, Tae Su et al. (2018) Native Top-Down Mass Spectrometry and Ion Mobility MS for Characterizing the Cobalt and Manganese Metal Binding of ?-Synuclein Protein. J Am Soc Mass Spectrom 29:1870-1880
Li, Huilin; Nguyen, Hong Hanh; Ogorzalek Loo, Rachel R et al. (2018) An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes. Nat Chem 10:139-148
Chang, Chungyu; Amer, Brendan R; Osipiuk, Jerzy et al. (2018) In vitro reconstitution of sortase-catalyzed pilus polymerization reveals structural elements involved in pilin cross-linking. Proc Natl Acad Sci U S A 115:E5477-E5486
Lermyte, Frederik; Valkenborg, Dirk; Loo, Joseph A et al. (2018) Radical solutions: Principles and application of electron-based dissociation in mass spectrometry-based analysis of protein structure. Mass Spectrom Rev 37:750-771
Susa, Anna C; Lippens, Jennifer L; Xia, Zijie et al. (2018) Submicrometer Emitter ESI Tips for Native Mass Spectrometry of Membrane Proteins in Ionic and Nonionic Detergents. J Am Soc Mass Spectrom 29:203-206
McConnell, Scott A; Amer, Brendan R; Muroski, John et al. (2018) Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae. J Am Chem Soc 140:8420-8423
Cleary, Sean P; Li, Huilin; Bagal, Dhanashri et al. (2018) Extracting Charge and Mass Information from Highly Congested Mass Spectra Using Fourier-Domain Harmonics. J Am Soc Mass Spectrom :
Zimmer, Richard K; Ferrier, Graham A; Kim, Steven J et al. (2017) Keystone predation and molecules of keystone significance. Ecology 98:1710-1721

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