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 especially diseases warrants a practical and sensitive method for their study and their structural elucidation. The superior sensitivity of mass spectrometry and the special ability of ESI to transfer solution-phase molecules to their gas-phase ionized counterparts without disrupting covalent bonds and maintaining weak non-covalent interactions are key for the study of protein complexes. ESI-MS can measure proteins and complexes in aqueous solution at near neutral pH, i.e., native MS. Moreover, top-down MS, the direct fragmentation of intact gas-phase large molecules to generate structure-informative product ions, has value for the structural characterization of protein complexes. Advanced techniques will be developed to facilitate the characterization of non-covalent protein assemblies. Novel methodologies and tools will be developed to improve the applicability of native MS. A critical barrier to membrane protein characterization by MS will be addressed. Methods to improve the efficiency and to expand the applicability of top-down MS for determining the binding sites of small molecule ligands on protein targets, and for elucidating the binding interface between large macromolecules will be developed. Enhancing multiple charging generated by ESI increases the efficiency for top-down MS and native MS. Combining top-down MS and hydrogen-deuterium exchange can deliver important structural information for protein complexes, such a protein-DNA/RNA complexes. The ability to enhance ESI charging for a variety of analytical platforms for MS will increase the applicability of ESI-MS. Top-down MS of protein-ligand and protein-protein complexes should deliver information on ligand binding location and protein-protein interfaces in a rapid manner that can be useful for biological systems of importance in biology, medicine, and human health. In the development of novel inhibitors, localization of their sites of interaction on the target of interest is a critical inital step in the drug discovery process. Our work to develop new methods for directly determining ligand binds sites may lead to a new strategy for screening potential drugs. Improvements in MS-based technologies can advance our views of how proteins and protein machines drive biology. Native MS will deliver important data that can be integrated with data derived from other biophysical techniques to generate 3D structural models to address relevant biological questions.

Public Health Relevance

We aim to develop more robust analytical technologies that can provide structural information for biologically important proteins, such as proteins involved i neurodegenerative disorders (e.g., Alzheimer's disease, Parkinson's disease) and bacterial infections. New methodologies to determine binding sites of drugs, other small molecules, proteins, and nucleic acids to targeted proteins will be developed. The information provided by our methods 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 #
5R01GM103479-11
Application #
8901220
Study Section
Enabling Bioanalytical and Imaging Technologies Study Section (EBIT)
Program Officer
Sheeley, Douglas
Project Start
2004-07-15
Project End
2018-06-30
Budget Start
2015-07-01
Budget End
2016-06-30
Support Year
11
Fiscal Year
2015
Total Cost
$331,797
Indirect Cost
$106,797
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

Showing the most recent 10 out of 55 publications