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Hydrogen/Deuterium Exchange, Supercharging and Top-down FT-ICR Mass Spectrometry of Biomolecules Method Development, Optimization and Applications

Title: Hydrogen/Deuterium Exchange, Supercharging and Top-down FT-ICR Mass Spectrometry of Biomolecules Method Development, Optimization and Applications.
Name(s): Valeja, Santosh Gurmukhdas, author
Marshall, Alan G., professor directing dissertation
Blaber, Michael, university representative
Roper, Michael G., committee member
Stagg, Scott M., 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: Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) provides the highest mass resolving power and mass measurement accuracy for confidant identification of biomolecules such as proteins and peptides. Chapter 1 briefly provides introductory information on FT-ICR MS, description of the 14.5 T and 9.4 T FT-ICR MS instruments in our laboratory and basics of liquid chromatographic separation, electrospray ionization (ESI), hydrogen/deuterium exchange (HDX) technique. The last part of this chapter gives an overview of the bottom-up and top-down approaches for protein identification and characterization and finally, tandem mass spectrometry methods used for dissociation of biomolecules. HDX monitored by MS is an important non-perturbing tool to study protein structure and protein-protein interactions. However, water in the reversed-phase liquid chromatography mobile phase leads to back-exchange of D for H during chromatographic separation of proteolytic peptides following H/D exchange, resulting in incorrect identification of fast-exchanging hydrogens as unexchanged hydrogens. Previously, fast HPLC and supercritical fluid chromatography have been shown to decrease back-exchange by our laboratory. Chapter 2 shows that replacement of up to 40% of the water in the LC mobile phase by the modifiers, dimethylformamide (DMF) and N-methylpyrrolidone (NMP) (i.e., polar organic modifiers that lack rapid exchanging hydrogens), significantly reduces back-exchange. On-line LC micro-ESI FT-ICR MS resolves overlapped proteolytic peptide isotopic distributions, allowing for quantitative determination of the extent of back-exchange. The DMF modified solvent composition also improves chromatographic separation while reducing back-exchange relative to conventional solvent. This work has been published in J. Am. Soc. Mass Spectrom., 2012, 23, 699-707. ESI produces multiply charged ions, thereby lowering the mass-to-charge ratio for peptides and small proteins to a range readily accessed by trapped-ion (quadrupole, orbitrap, and ion cyclotron resonance (ICR)) mass analyzers (m/z = 400-2,000). For Fourier transform mass analyzers (orbitrap and ICR), higher charge also improves signal-to-noise ratio, mass resolution, and mass accuracy. Addition of m-nitrobenzyl alcohol (m-NBA) or sulfolane has previously been shown to increase the charge states of proteins. Moreover, polar aprotic DMF improves chromatographic separation of proteolytic peptides for mass analysis of solution-phase protein HDX for improved (78-96%) sequence coverage. In chapter 3, we show that addition of each of various modifiers (DMF, thiodiglycol, dimethylacetamide, dimethylsulfoxide, and N-methylpyrrolidone) can significantly increase the charge states of proteins up to 78 kDa. Moreover, incorporation of the same modifiers into reversed-phase liquid chromatography solvents improves sensitivity, charging, and chromatographic resolution for intact proteins. This work has been published in Anal. Chem., 2010, 78, 7515 - 7519. In chapter 4, we investigate the extent of charging of different peptides and polyethylene glycols electrosprayed from different solutions with various modifiers to gain more insight into the supercharging mechanism. Small peptides without significant secondary structure exhibits higher sensitivity and shifts to higher charge states with DMSO. Also, DMSO increases charge and S/N for PEG derivatives as well. Furthermore, we discuss the preliminary results of ESI droplet size measurement with phase Doppler particle analyzer (PDPA) and also review all possible reasons that could potentially contribute to supercharging. Chapter 5 talks about the top-down electron capture dissociation (ECD) characteristics of multiply charged molecular ions of various proteins formed by the incorporation of supercharging reagents. Here, we show that addition of modifier and ECD of higher charge states can significantly increase the number of structurally informative backbone cleavages and diagnostic product ions. Moreover, though ECD of higher charge states improves protein sequencing, the sequence coverage for cytochrome c gradually decreases beyond a particular charge state. Also, there is no significant difference in the fragmentation of same charge state with various modifiers. ECD of supercharged ions can be anticipated to improve top-down sequencing and characterization especially for high molecular weight protein complexes. Previously, the highest-mass protein for which FT-ICR unit mass resolution had been obtained was 115 kDa at 7 T. In chapter 6, we present baseline resolution for an intact 147.7 kDa monoclonal antibody (mAb), by prior dissociation of non-covalent adducts, optimization of detected total ion number, and optimization of ICR cell parameters to minimize space charge shifts, peak coalescence, and destructive ion cloud Coulombic interactions. The resultant long ICR transient lifetime (as high as 20 s) results in magnitude-mode mass resolving power of ~420,000 at m/z 2,593 for the 57+ charge state (the highest mass for which baseline unit mass resolution has been achieved), auguring for future characterization of even larger intact proteins and protein complexes by FT-ICR MS. We also demonstrate up to 80% higher resolving power by phase correction to yield an absorption-mode mass spectrum. This work has been published in Anal. Chem., 2011, 83, 8391-8395. The dissociation efficiency of intact proteins decreases as the molecular size and structural complexity increases due to the non-covalent interactions that stabilize the higher order structures of intact protein. Chapter 7 is a short review that describes the challenges and improvements in the direct top-down dissociation of large intact proteins (>60 kDa). Various innovative methods of collisional activated dissociation (CAD) such as prefolding dissociation, octopole-CAD, multipole-storage-assisted dissociation (MSAD) etc and electron driven dissociation techniques coupled with mostly ultra-high resolution FT instruments are covered. The appendixes include the manuscripts I have published during my Ph.D. Appendix A is the paper that covers the method development and optimization to decrease back-exchange in HDX by incorporation of aprotic modifiers. Appendix B is a paper we published in Analytical Chemistry, covering enhanced charging and improved chromatographic separation of intact proteins by addition of new organic modifiers. Appendix C is a publication that describes record unit mass baseline resolution for an intact 148 kDa monoclonal antibody by FT-ICR MS in collaboration with Pfizer, Inc.
Identifier: FSU_migr_etd-8472 (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 25, 2013.
Bibliography Note: Includes bibliographical references.
Advisory Committee: Alan G. Marshall, Professor Directing Dissertation; Michael Blaber, University Representative; Michael G. Roper, Committee Member; Scott M. Stagg, Committee Member.
Subject(s): Chemistry
Persistent Link to This Record:
Owner Institution: FSU

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Valeja, S. G. (2013). Hydrogen/Deuterium Exchange, Supercharging and Top-down FT-ICR Mass Spectrometry of Biomolecules Method Development, Optimization and Applications. Retrieved from