You are here

Hydrogen/Deuterium Exchange Coupled with FT-ICR MS

Title: Hydrogen/Deuterium Exchange Coupled with FT-ICR MS: Instrumentation, Software Development and Applications.
Name(s): Zhang, Qian, author
Marshall, Alan G., professor directing dissertation
Blaber, Michael, university representative
Stagg, Scott M., committee member
Roper, Michael G., committee member
Department of Chemistry and Biochemistry, degree granting department
Florida State University, degree granting institution
Type of Resource: text
Genre: Text
Issuance: monographic
Date Issued: 2013
Publisher: Florida State University
Place of Publication: Tallahassee, Florida
Physical Form: computer
online resource
Extent: 1 online resource
Language(s): English
Abstract/Description: Structural biology is a field studying the relationship between structure and function of macro biomolecules, including proteins. Proteins with numerous different functions are synthesized by recruiting amino acids into a linear polypeptide chain. The linear protein sequence, determined by a gene, can fold into higher level structures. For a given protein, correct structure/conformation is important to maintain its proper biological function. Several biophysical tools such as X-ray crystallography, NMR and cryo-EM can be used to investigate protein structure. However, each method has its limitations and usually complementary methods are required to answer a complicated biological question. Hydrogen/deuterium exchange coupled with mass spectrometry (HDX MS) has become a informative tool to study solution phase protein conformation. High-resolution mass analysis (such as Fourier transform ion cyclotron resonance mass spectrometer) is particularly advantageous for the HDX MS method, by accurately assigning the peptide fragments after protease digestion and in resolving extensive overlap of peptide isotopic distributions, both before and after HDX MS. Chapter 1 is started by introducing the theory of the FT-ICR MS and the actual instrumentation set-up of the Hybrid LTQ 14.5 T FT-ICR Mass Spectrometer. An introduction of several biophysical methods to study structural biology is also covered, followed by the examples of the usage of mass spectrometry in this field. The last portion of this chapter gives an overview of the HDX MS method, its principle, experimental strategy and the historical applications.In Chapter 2, a new automation system is described as part of the method development effort for HDX-MS in my PhD career. The new system permits flexible modification of all HDX parameters, e.g., number of HDX reactions, reaction volume, injection volume, HDX duration, quench duration, digestion duration, and chromatography duration. Additional steps such as pre-HDX handling of protein sample, additional replicates, or post-digestion treatments can be easily added and automatically interlaced. Chapter also describes the software developments for HDX-MS data analysis, including several add-ons developed in Python: 'Peak list based data evaluation.', 'Compare HDX data for multiple protein states.' and 'HDX heat-map and modeling HDX results on 3D structure.' Chapter 3 describes the HDX-MS application on the epitope mapping problems. Three projects are described consequently. The first project is a 'proof of principle' study trying to establish the HDX-MS method on a known antigen-antibody system. The second and third projects are the applications of HDX-MS method on detecting unknown epitopes. The result of the first project is published in Analytical Chemistry (Zhang, Q. et al. Anal. Chem. 2011, 83, 7129-7136.). The manuscripts of the latter two projects are being submitted soon. Chapter 4 describes a collaborative project combining cryo-EM and HDX-MS to solve the structure of COPII cage. COPII vesicles are involved in transporting proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. A 12 Å resolution structure of the COPII cage, from which the tertiary structure of Sec13 and Sec31 is clearly identifiable, is generated by cryo-EM data. This structure and a homology model of the Sec13-31 are coupled with flexible fitting methods to create a reliable pseudo-atomic model of the COPII cage. Data from hydrogen/deuterium exchange mass spectrometry analysis is combined with this model to characterize four distinct contact regions at the vertices of the COPII cage. These results are published in Nature Structure Molecular Biology recently. Chapter 5 discusses the use of solution-phase HDX experiments to probe the allosteric effect of ATP on molecular chaperone GroEL. The ~800 kDa tetradecameric GroEL plays an essential role in the proper folding of many cellular proteins via an ATP-driven cycle of conformational changes. We capture the preceding yet pivotal step in its functional cycle by use of a non-hydrolysable ATP analog, ATPγS, to mimic the ATP-bound GroEL intermediate conformational state. Comparison of HDX-MS results for apo GroEL and GroEL-ATPγS enables the characterization of the nucleotide-regulated conformational changes throughout the entire protein with high sequence resolution. GroEL is by far the largest protein entity yet accessed by HDX-MS, and the results achieved here establish the groundwork for further HDX-MS characterization of such large complexes in solution. This work has been recently accepted by Scientific Reports. The appendices include the papers I have published during my graduate study. Appendix A is a publication that covers the collaboration work with Dr. Kenneth Roux (FSU department of biology) on the epitope mapping of food allergens. Appendix B is the paper we published in Nature Structure Molecular Biology recently, covering the COPII cage structure project.
Identifier: FSU_migr_etd-8096 (IID)
Submitted Note: A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy.
Degree Awarded: Spring Semester, 2013.
Date of Defense: February 18, 2013.
Bibliography Note: Includes bibliographical references.
Advisory Committee: Alan G. Marshall, Professor Directing Dissertation; Michael Blaber, University Representative; Scott M. Stagg, Committee Member; Michael G. Roper, Committee Member.
Subject(s): Chemistry
Persistent Link to This Record:
Owner Institution: FSU

Choose the citation style.
Zhang, Q. (2013). Hydrogen/Deuterium Exchange Coupled with FT-ICR MS: Instrumentation, Software Development and Applications. Retrieved from