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

1. 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.

2. 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.

3. 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.

4. 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

38. Hatvany, J. B.; Olsen, E. P.; Gallagher, E. S. Characterizing Theta-Emitter Generation for Use in Microdroplet Reactions. J. Am. Soc. Mass Spectrom. 2024, XX (XX), XXX-XXX. DOI: 10.1021/jasms.4c00262

37. Conforti, J. M.; Ziegler, A. M.; Worth, C. S.; Nambiar, A. M.; Bailey, J. T.; Taube, J. H.; Gallagher, E. S. Differences in Protein Capture by SP3 and SP4 Demonstrate Mechanistic Insights of Proteomics Cleanup Techniques. J. Proteome Res. 2024, 23 (9), 3877-3889. DOI: 10.1021/acs.jproteome.4c00206

36. Quintero, A. V.; Liyanage, O. T., Kim, H. J.; Gallagher, E. S. Characterizing the Dynamics of Solvated Disaccharides with In-Electrospray Ionization Hydrogen/Deuterium Exchange-Mass Spectrometry. Anal. Chem. 2024, 96 (31), 12649-12657. DOI: 10.1021/acs.analchem.4c01136

35. Cordes, M. S.; Gallagher, E. S. Molecular Dynamics Simulations of Native Protein Charging via Proton Transfer during Electrospray Ionization with Grotthuss Diffuse H3O+. Anal. Chem. 2024, 96 (10), 4146-4153. DOI: 10.1021/acs.analchem.3c05089

34. Hatvany, J. B.; Liyanage, O. T.; Gallagher, E. S. Effect of pH on In-Electrospray Hydrogen/Deuterium Exchange of Carbohydrates and Peptides. J. Am. Soc. Mass Spectrom. 2024, 35 (3), 441-448. DOI: 10.1021/jasms.3c00341

33. Gass, D. T.; Cordes, M. S.; Alberti, S. N.; Kim, H. J.; Gallagher, E. S. Evidence of H/D Exchange within Metal-Adducted Carbohydrates after Ion/Ion-Dissociation Reactions. J. Am. Chem. Soc. 2023, 145 (44), 23972-23985. DOI: 10.1021/jacs.3c05793

32. Edwards, A. N.; Blue, A. J., Conforti, J. M., Cordes, M. S.; Trakselis, M. A., Gallagher, E. S. Gas-phase stability and thermodynamics of ligand-bound, binary complexes of chloramphenicol acetyltransferase reveal negative cooperativity. Anal. Bioanal. Chem. 2023, 415 (25), 6201-6212. DOI: 10.1007/s00216-023-04891-5

31. Hatvany, J. B.; Gallagher, E. S. Hydrogen/deuterium exchange for the analysis of carbohydrates. Carbohydr. Res. 2023, 530, 108859. DOI: 10.1016/j.carres.2023.108859

30. Gass, D. T.; Quintero, A.V.; Hatvany, J.B.; Gallagher, E. S. Metal adduction in mass spectrometric analyses of carbohydrates and glycoconjugates. Mass Spectrom. Rev. 2024, 43 (1), 615-659. DOI: 10.1002/mas.21801

29. Calixte, E. I.; Liyanage, O. T.; Gass, D. T.; Gallagher, E. S. Formation of Carbohydrate-Metal Adducts from Solvent Mixtures during Electrospray: A Molecular Dynamics and ESI-MS Study. J. Am. Soc. Mass Spectrom. 2021, 32 (12), 2738-2745. DOI: 10.1021/jasms.1c00179

28. Edwards, A. N.; Tran, H. M.; Gallagher, E. S. Propagating Error through Traveling-Wave Ion Mobility Calibration. J. Am. Soc. Mass Spectrom. 2021, 32 (11), 2621-2630. DOI: 10.1021/jasms.1c00144

27.     Liyanage, O. T.; Quintero, A. V.; Hatvany, J. B.; Gallagher, E. S. Distinguishing Carbohydrate Isomers with Rapid Hydrogen/Deuterium Exchange-Mass Spectrometry. J. Am. Soc. Mass. Spectrom. 2020, 32 (1), 152-156. DOI: 10.1021/jasms.0c00314

26.     Kim, H. J.; Gallagher, E. S. Achieving Multiple Hydrogen/Deuterium Exchange Timepoints of Carbohydrate Hydroxyls using Theta-Electrospray Emitters. Analyst. 2020, 145 (8), 3056-3063. DOI:  10.1039/D0AN00135J

25.     Calixte, E. I.; Liyanage, O. T.; Kim, H. J.; Ziperman, E. D.; Pearson, A. J.; Gallagher, E. S. Release of Carbohydrate-Metal Adducts from Electrospray Droplets: Insight into Glycan Ionization by Electrospray. J. Phys. Chem. B. 2019, 124 (3), 479-486. DOI: 10.1021/acs.jpcb.9b10369

24. Ziperman, E. D.; Calixte, E. I.; McCutcheon, M., Pulipaka, S., Gallagher, E. S. Examining the Accuracy of Monosaccharide Structures Calculated with Density Functional Theory by Experimental Comparisons. FASEB J. 2019, 33 (S1), 642.9-642.9. DOI: 10.1096/fasebj.2019.33.1_supplement.642.9

23.     Liyanage, O. T.; Seneviratne, C. A.; Gallagher, E. S. Applying an Internal Standard to Improve the Repeatability of In-Electrospray H/D Exchange of Carbohydrate-Metal Adducts. J. Am. Soc. Mass Spectrom. 2019, 30, 1368-1372. DOI: 10.1007/s13361-019-02153-2

22. Pearson, A. J.; Liedig, C.; Gallagher, E. S. Expression and Purification of Soluble, Glycosylated T-cell Receptors in Chinese Hamster Ovary Cells. FASEB J. 2019, 33 (S1), 472.6-472.6. DOI: 10.1096/fasebj.2019.33.1_supplement.472.6

21.      Liyanage, O. T.; Brantley, M. R.; Calixte, E. I.; Solouki, T.; Shuford, K. L.; Gallagher, E. S. Characterization of Electrospray Ionization (ESI) Parameters on In-ESI Hydrogen/Deuterium Exchange of Carbohydrate-Metal Ion Adducts. J. Am. Soc. Mass Spectrom. 2019, 30 (2), 235–247. DOI: 10.1007/s13361-018-2080-1

20.     Pearson, A. J.; Gallagher, E. S. 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, H. J.; Liyanage, O. T.; Mulenos, M. R.; Gallagher, E. S. Mass Spectral Detection of Forward- and Reverse-Hydrogen/Deuterium Exchange Resulting from Residual Solvent Vapors in Electrospray Sources. J Am Soc Mass Spectrom. 2018, 29 (10), 2030-2040. DOI: 10.1007/s13361-018-2019-6

Prior Work by Dr. ES Gallagher

18. Anderson, K. W.; Bergonzo, C.; Scott, K.; Karageorgos, I. L.; Gallagher, E. S.; Tayi, V. S.; Butler, M.; Hudgens, J. W. HDX-MS and MD simulations provide evidence for stabilization of the IgG1-FcyR1a (CD64a) immune complex through intermolecular glycoprotein bonds. J. Mol. Bio. 2022, 434 (2), 167391. DOI: 10.1016/j.jmb.2021.167391

17. Anderson, K. W.; Scott, K.; Karageorgos, I. L.; Gallagher, E. S.; Tayi, V. S.; Butler, M.; Hudgens, J. W. Dataset from HDX-MS Studies of IgG1 Glycoforms and Their Interactions with the FcyR1a (CD64) Receptor. J. Res. Natl. Inst. Stand. Technol. 2021, 126, 126010. DOI: 10.6028/jres.126.010

16.       Hudgens, J. W.; Gallagher, E. S.; Karageorgos, I.; Anderson, K. W.; Filliben, J. J.; 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, 91 (11), 7336-7345. DOI: 10.1021/acs.analchem.9b01100

15. Hudgens, J. W.; Gallagher, E. S.; Karageorgos, I.; Anderson, K. W. ; Huang, RY-C.; Chen, G.; Bau-Assaf, G. M., 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, K. E.; Condarcure, A. M.; Sutton, C. T.; Baker, C. A.; Gallagher, E. S.; Bright, L. K.; Aspinwall, C. A. Rapid formation of polymer frits in fused silica capillaries using spatially defined thermal free-radical initiated polymerization. Sep Sci plus. 2018, 1 (11), 753-758. DOI:10.1002/sscp.201800126

13.   Anderson, K. W.; Gallagher, E. S.; Hudgens, J. W. Automated removal of phospholipids from membrane proteins for H/D exchange mass spectrometry workflows. Anal Chem. 2018, 90 (11), 6409-6412. DOI: 10.1021/acs.analchem.8b00429

12.   Tyukhtenko, S.; Rajarshi, G.; Karageorgos, I.; Zvonok, N.; Gallagher, E. S.; Huang, H.; Vemuri, K.; Hudgens, J. W.; Ma, X.; Nasr, M. L.; Pavlopoulos, S.; Makriyannis, A. Effects of Distal Mutations on the Structure, Dynamics and Catalysis of Human Monoacylglycerol Lipase. Sci Rep. 2018, 8 (1), 1719. DOI: 10.1038/s41598-017-19135-7

11.   Karageorgos, I.; Gallagher, E. S.; Galvin, C.; Gallagher, D. T.; Hudgens, J. W. Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671. Biologicals. 2017, 50, 27-34. DOI: 10.1016/j.biologicals.2017.09.005

10.   Mazur, S. J.; Gallagher, E. S.; Debnath, S.; Durell, S. R.; Anderson, K. W.; Miller Jenkins, L. M.; Appella, E.; Hudgens, J. W. Conformational Changes in Active and Inactive States of Human PP2Cα Characterized by Hydrogen/Deuterium Exchange-Mass Spectrometry. Biochemistry 2017, 56 (21), 2676-2689. DOI: 10.1021/acs.biochem.6b01220

9.   Gallagher, E. S.; Hudgens, J. W. Chapter 14 - Mapping Protein-Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry. Isotope Labeling of Biomolecules – Methods in Enzymology, 2016, 566, 357-404. DOI: 10.1016/bs.mie.2015.08.010

8.   Marino, J. P.; Brinson, R. G.; Hudgens, J. W.; Ladner, J. E.; Gallagher, D. T.; Gallagher, E. S.; Arbogast, L. W.; Huang, RY-C. Chapter 2 - Emerging Technologies to Assess the Higher-Order Structure of Monoclonal Antibodies. State-of-the-Art & Emerging Technologies for Therapeutic Monoclonal Antibody Characterization. 2015, 3, 17-43. DOI: 10.1021/bk-2015-1202.ch002

7.   Gallagher, E. S.; Adem, S. M.; Baker, C. A.; Ratnayaka, S. N.; Jones, I. W.; Hall, H. K. Jr; Saavedra, S. S.; Aspinwall, C. A. Highly Stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography. J Chromatogr A. 2015, 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. 2016. Systems and methods of preparing stabilized lipid assemblies. World Intellectual Property Organization Patent Application WO 2,016,004,029 A1 filed June 30, 2015, issued January 7, 2016.

5.     Aspinwall, C. A.; Wang, J.; Sandy, K.; Saavedra, S. S.; Baker, C.; Gallagher, E. S.; Boying, L. 2021. Stabilized vesicle-functionalized microparticles for chemical separations and rapid formation of polymer frits in silica capillaries using spatially-defined thermal polymerization. United States Patent Application US 20,210,088,509 A1, filed August 10, 2015, issued March 25, 2021.

4.   Johnson, G. M.; Chozinski, T. J.; Gallagher, E. S.; Aspinwall, C. A.; Miranda, K. M. Glutathione sulfinamide serves as a selective, endogenous biomarker for nitroxyl after exposure to therapeutic levels of donors. Free Radic Biol Med. 2014, 76, 299-307. DOI: 10.1016/j.freeradbiomed.2014.07.022

3.   Gallagher, E. S.; Adem, S. M.; Bright, L. K.; Calderon, I. A. C.; Mansfield, E.; Aspinwall, C. A. Hybrid Phospholipid Bilayer Coatings for Separations of Cationic Proteins in Capillary Zone Electrophoresis. Electrophoresis. 2014, 35 (8), 1099-1105. DOI: 10.1002/elps.201300537

2.   Gallagher, E. S.; Mansfield, E.; Aspinwall, C. A. Stabilized Phospholipid Membranes in Chromatography: Towards Membrane Protein-Functionalized Stationary Phases. Anal Bioanal Chem. 2014, 406 (9-10), 2223-2229. DOI: 10.1007/s00216-013-7545-2

1.   Gallagher, E. S.; Comi, T. J.; Braun, K.; Aspinwall, C. A. Online Photolytic Optical Gating of Caged Fluorophores Utilizing an Ultraviolet Light Emitting Diode. Electrophoresis. 2012, 33 (18), 2903-2910, DOI: 10.1002/elps.201200279

Current Funding