Research
The Gallagher Group is a bioanalytical research group in the Department of Chemistry and Biochemistry at Baylor University. Research in the group involves method development, but also explores fundamental processes in electrospray ionization and mass spectrometry as well as analysis of biological samples. We currently have 4 major areas where we contribute to science
Fundamentals of Carbohydrate Ionization and Analysis by Mass Spectrometry.
It is common for carbohydrates to be characterized by mass spectrometry-based techniques, such as ion mobility and tandem mass spectrometry. For these experiments, electrospray ionization (ESI) is often used to produce ions. As carbohydrates transition from solvated analytes in ESI droplets to gas-phase ions in positive-ion mode, they often coordinate to metal ions with the properties of the metal affecting both the three-dimensional, gas-phase conformations and properties of the carbohydrate-metal adducts. Our group has used a combination of molecular dynamics (MD) simulations and ESI mass spectrometry to explore carbohydrate-metal-ion adduct formation during ESI. This work has provided molecular insights to describe how ESI alters carbohydrate conformations and generates the adducts that are detected by mass spectrometry. This work is significant because it includes the first investigations to illustrate the mechanisms of carbohydrate ionization. In addition to the fundamentals of ESI, we are examining how metal-ion adducts affect carbohydrate analyses by mass spectrometry-based techniques, including tandem mass spectrometry.
Native Mass Spectrometry of Proteins.
Native mass spectrometry and ion mobility are powerful tools that are being used to address many questions related to structural biology. However, measurements of ion mobility and reports of collisional cross section (CCS), the three-dimensional, gas-phase structures of protein analytes, are limited by assumptions and experimental uncertainties. Therefore, we have developed a method to propagate error through CCS calibrations of traveling-wave ion mobility instruments, which improves agreement with reported CCS values from the literature. This provides a more robust assessment of uncertainty related to gas-phase structures, enabling comparisons across different ion mobility instruments and by different analytical techniques.
Hydrogen/Deuterium Exchange-Mass Spectrometry for Analysis of Glycan Structure.
Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is an established tool for monitoring the structure, dynamics, and noncovalent interactions of proteins and protein-ligand complexes. However, the traditional, solution-phase HDX-MS methods cannot be applied to carbohydrates due to the rapid exchange rates of hydroxyls. Our lab has focused on the development and application of rapid HDX-MS methods to characterize carbohydrate conformations. Thus far, we have addressed this goal by developing and utilizing in-electropray ionization HDX-MS for rapid labeling of carbohydrates. To date, we have shown that these rapid HDX methods sample solvated carbohydrates. We have optimized rapid-labeling HDX methods and developed an internal standard to control day-to-day variability. We have developed methods to expand the HDX labeling time. We have also applied these techniques to conformational analyses of carbohydrate isomers. Our lab recently received the Ron Hites Award for our publication describing the application of HDX to carbohydrate isomers.
Proteomics.
Proteomics is an increasingly popular tool for proteoform analysis due to the variety of sequence variations, isoforms, and post translational modifications in proteins. Full knowledge of the proteome is essential to identify specific biomarkers for diseases and molecular targets for drug discovery. Sample preparation for complex protein mixtures is typically complicated due to unwanted detergents and buffers needed to extract proteins from cells prior to MS analysis. We have established a working protocol for proteoform analysis using solvent precipitation, single-pot, solid-phase-enhanced sample preparation (SP4) combined with a detergent-based digestion and peptide clean-up. A manuscript for this work is currently in preparation and was presented at a recent conference due to the enhanced hydrophobic peptide yield identified with our optimization of SP4. This protocol can be applied to the analysis of post-translational modifications, protein validation, and protein physiological role, as needed by collaborating groups. Currently, we are focusing on methylation, oxidation, and hydrogen/deuterium exchange modifications in proteins, and plan on expanding our proteomics platform towards more collaborations in the future.
Recent Publications
33. Cordes MS, Gallagher ES. Molecular Dynamics Simulations of Native Protein Charging via Proton Transfer during Electrospray Ionization with Grotthuss Diffuse H3O+ Analytical Chemistry 2024 DOI 10.1021/acs.analchem.3c05089
32. Hatvany JB, Liyanage OT, Gallagher ES. Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides. J. Am. Soc. Mass Spectrom. 2024 DOI 10.1021/jasms.3c00341
31. Gass DT, Cordes MS, Alberti SN, Kim HJ, Gallagher ES. Evidence of H/D Exchange within Metal-Adducted Carbohydrates after Ion/Ion-Dissociation Reactions. Journal of the American Chemical Society 2023 doi: 10.1021/jacs.3c05793
30. Edwards AN, Blue AJ, Conforti JM, Cordes MS, Trakselis MA, Gallagher ES. Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity. Analytical and Bioanalytical Chemistry 2023 doi: 10.1007/s00216-023-04891-5
29. Hatvany JB, Gallagher ES. Hydrogen/deuterium exchange for the analysis of carbohydrates. Carbohydrate Research 2023 doi: 10.1016/j.carres.2023.108859
28. Gass DT, Quintero AV, Hatvany JB, Gallagher ES. Metal adduction in mass spectrometric analyses of carbohydrates and glycoconjugates. Mass Spectrometry Reviews 2022 Aug; doi: 10.1002/mas.21801
27. Calixte EI, Liyanage OT, Gass DT, Gallagher ES. Formation of carbohydrate-metal adducts from solvent mixtures during electrospray ionization. J. Am. Soc. Mass Spectrom. 2021 Dec; 32(12): 2738-2745. doi: 10.1021/jasms.1c00179.
26. Edwards AN, Tran HM, Gallagher ES. Propagating Error through Traveling-Wave Ion Mobility Calibration. J. Am. Soc. Mass Spectrom. 2021 Nov; 32(11): 2621-2630. doi: 10.1021/jasms.1c00144.
25. Liyanage OT, Quintero AV, Hatvany JB, Gallagher ES. Distinguishing Carbohydrate Isomers with Rapid Hydrogen/Deuterium Exchange – Mass Spectrometry. J Am Soc Mass Spectrom. 2021 Jan; 32(1): 152-156. https://doi.org/10.1021/jasms.0c00314
24. Kim HJ, Gallagher ES. Achieving Multiple Hydrogen/Deuterium Exchange Timepoints of Carbohydrate Hydroxyls Using Theta-Electrospray Emitters. Analyst 2020; 145: 3056-3063. https://doi.org/10.1039/D0AN00135J.
23. Calixte EI, Liyanage OT, Kim HJ, Ziperman ED, Pearson AJ, Gallagher, E.S. Release of carbohydrate-metal adducts from electrospray droplets: Insight into glycan ionization by electrospray. J Phys Chem B. 2020 Jan; 124(3): 479-486. doi: 10.1021/acs.jpcb.9b10369.
22. Liyanage OT, Seneviratne, CA, Gallagher, ES. Applying an internal standard to improve the repeatability of in-electrospray H/D exchange of carbohydrate-metal adducts. J Am Soc Mass Spectrom. 2019 Aug; 30(8): 1368-1372. doi: 10.1007/s13361-019-02153-2.
21. Liyanage OT, Brantley MR, Calixte EI, Solouki T, Shuford KL, Gallagher ES. Characterization of Electrospray Ionization (ESI) Parameters for In-ESI Hydrogen/Deuterium Exchange of Carbohydrate-Metal Ion Adducts. J. Am. Soc. Mass Spectrom. 2019 Feb; 30(2): 235-247. doi. 10.1007/s13361-018-2080-1.
20. Pearson AJ, Gallagher ES. Overview of Characterizing Cancer Glycans with Lectin-Based Analytical Methods. Methods Mol Biol. 2019; 1928: 389-408. doi: 10.1007/978-1-4939-9027-6_20.
19. Kim HJ, Liyanage OT, Mulenos MR, Gallagher ES. Mass Spectral Detection of Forward- and Reverse- Hydrogen/Deuterium Exchange Resulting from Residual Solvent Vapors in Electrospray Sources. J Am Soc Mass Spectrom. 2018 Jul; 29(10): 2030-2040. doi. 10.1007/s13361-018-2019-6.
Prior Work by Dr. ES Gallagher
18. Anderson KW, Bergonzo C, Scott K, Karageorgos IL, Gallagher ES, Tayi VS, Butler M, Hudgens JW. HDX-MS and MD simulations provide evidence for stabilization of the IgG1-FcyR1a (CD64a) immune complex through intermolecular glycoprotein bonds. J. Mol. Bio. 2022 Jan; 434(2): 167391. doi: 10.1016/j.jmb.2021.167391.
17. Anderson KW, Scott K, Karageorgos IL, Gallagher ES, Tayi VS, Butler M, Hudgens JW. Dataset from HDX-MS Studies of IgG1 Glycoforms and Their Interactions with the FcyR1a (CD64) Receptor. J. Res. Natl. Inst. Stand. Technol. 2021, 126, page N/A. doi:10.6028/jres.126.010.
16. Hudgens JW, Gallagher ES, Karageorgos I, Anderson KW, Filliben JJ, Huang RY-C, Chen G, et. al. Interlaboratory Comparison of Hydrogen-Deuterium Exchange Mass Spectrometry Measurements of the Fab fragment of NISTmAb. Anal Chem. 2019 Jun; 91(11): 7336-7345. https://doi.org/10.1021/acs.analchem.9b01100.
15. Hudgens JW, Gallagher ES, Karageorgos I, Anderson KW, Huang RY-C, Chen G, Bau-Assaf GM, et. al. Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Centroid Data Measured between 3.6 C and 25.4 C for the Fab Fragment of NISTmAb. J. Res. Natl. Inst. Stand. Technol. 2019, 124, 1-7. doi: 10.6028/jres.124.009.
14. Sandy KE, Condarcure AM, Sutton CT, Baker CA, Gallagher ES, Bright LK, Aspinwall CA. Rapid formation of polymer frits in fused silica capillaries using spatially defined thermal free-radical initiated polymerization. Sep Sci plus. 2018 Nov; 1(11): 753-758. doi:10.1002/sscp.201800126.
13. Anderson KW, Gallagher ES, Hudgens JW. Automated removal of phospholipids from membrane proteins for H/D exchange mass spectrometry workflows. Anal Chem. 2018 Jun; 90(11): 6409-6412. doi: 10.1021/acs.analchem.8b00429.
12. Tyukhtenko S, Rajarshi G, Karageorgos I, Zvonok N, Gallagher ES, Huang H, Vemuri K, Hudgens JW, Ma X, Nasr ML, Pavlopoulos S, Makriyannis A. Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase. Sci Rep. 2018 Jan; 8(1): 1719. doi: 10.1038/s41598-017-19135-7.
11. Karageorgos I, Gallagher ES, Galvin C, Gallagher DT, Hudgens JW. Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671. Biologicals. 2017 Nov; 50: 27-34. doi: 10.1016/j.biologicals.2017.09.005.
10. Mazur SJ, Gallagher ES, Debnath S, Durell SR, Anderson KW, Miller Jenkins LM, Appella E, Hudgens JW. Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry. Biochemistry 2017 Mar; 56(21): 2676-2689. doi:10.1021/acs.biochem.6b01220.
9. Gallagher ES, Hudgens JW. “Mapping Protein-Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry.” in Isotope Labeling of Biomolecules – Applications Vol. 566 Methods in Enzymology (ed Zvi Kelman) Ch. 14, 357-404 (Academic Press, 2016). doi: 10.1016/bs.mie.2015.08.010.
8. Marino JP, Brinson RG, Hudgens JW, Ladner JE, Gallagher DT, Gallagher ES, Arbogast LW, Huang RY-C. “Emerging Technologies to Assess the Higher-Order Structure of Monoclonal Antibodies” in State-of-the-Art & Emerging Technologies for Therapeutic Monoclonal Antibody Characterization. Vol. 3: Defining the Next Generation of Analytical and Biophysical Techniques. (eds J.E. Shiel, D.L. Davis, and O.V. Borisov) Ch. 2, (American Chemical Society, 2015). doi: 10.1021/bk-2015-1202.ch002
7. Gallagher ES, Adem SM, Baker CA, Ratnayaka SN, Jones IW, Hall HK Jr, Saavedra SS, Aspinwall CA. Highly Stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography. J Chromatogr A. 2015 Mar; 1385: 28-34. doi: 10.1016/j.chroma.2015.01.052.
6. Saavedra, S.S.; Aspinwall, C.A.; Ratnayaka, S.N.; Gallagher, E.S.; Bright, L. Systems and methods of preparing stabilized lipid assemblies. Patent Application WO2016004029 A1 filed June 30, 2015.
5. Aspinwall, C.A.; Wang, J.; Sandy, K.; Saavedra, S.S.; Baker, C.; Gallagher, E.S. Stabilized vesicle-functionalized microparticles for chemical separations and rapid formation of polymer frits in silica capillaries using spatially defined thermal polymerization. U.S. Patent Application US20180224438A1 (also: WO2017027523A1) filed August 10, 2015.
4. Johnson GM, Chozinski TJ, Gallagher ES, Aspinwall CA, Miranda KM. Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors. Free Radic Biol Med. 2014 Nov; 76: 299-307. doi: 10.1016/j.freeradbiomed.2014.07.022.
3. Gallagher ES, Adem SM, Bright LK, Calderon IAC, Mansfield E, Aspinwall CA. Hybrid Phospholipid Bilayer Coatings for Separations of Cationic Proteins in Capillary Zone Electrophoresis. Electrophoresis 2014 Apr; 35(8): 1099-1105. doi: 10.1002/elps.201300537.
2. Gallagher ES, Mansfield E, and Aspinwall CA. Stabilized Phospholipid Membranes in Chromatography: Towards Membrane Protein-Functionalized Stationary Phases. Anal Bioanal Chem. 2014 Apr; 406(9-10): 2223-2229. doi:10.1007/s00216-013-7545-2.
1. Gallagher ES, Comi TJ, Braun K, Aspinwall CA. Online Photolytic Optical Gating of Caged Fluorophores Utilizing an Ultraviolet Light Emitting Diode. Electrophoresis 2012 Sep; 33(18): 2903-2910, doi: 10.1002/elps.201200279.
