Baylor University

Assessing the Biomolecular Structures That Result From Electrospray Ionization - Project Summary / Abstract Glycans, Heterogeneous Polymers of Carbohydrates, Interact With Proteins to Initiate a Multitude of Biologi- Cal Processes, Including Molecular Recognition, Cellular Signaling, and Host-microbe Interactions. Mass Spec- Trometry (MS) Methods Have Become Powerful Tools for Characterizing the Structures of Glycans and Their Inter- Actions With Proteins. Electrospray Ionization (ESI) Is a Common Ionization Method for Transferring These Bio- Molecules From Solution to the Gas-phase for MS Analysis. However, Both Glycans and Proteins Exhibit Struc- Tural Changes During Esi. Thus, There Is a Critical Need to Understand How Biomolecular Structures Are Modified During and After the Esi Process to Determine (1) How Researchers Can Deduce Solvated Structures From These Techniques and (2) How Analytical Methods Can Be Improved. to Address This Challenge, the Gallagher Lab Uses a Combination of Molecular Dynamics (MD) Simulations and MS Methods to Develop a Fundamental, Molecular Perspective of Esi. the Gallagher Lab Performs MD Simulations of Esi to Observe Carbohydrate Ionization Using Metal Ions as Charge Carriers. However, to More Accurately Model Protein Ionization, the Gallagher Lab Is Developing Methods to Simulate Protonation During Esi. These Methods Will Be Further Developed to Examine Analyte Ionization by Deprotonation in Negative-ion Mode, and Then Applied to Achieve a Molecular Perspective on the Ionization of Glycans and Proteins. in Paral- Lel to the MD Simulations, the Gallagher Lab Will Perform MS Analysis of Metal-adducted Glycans. Glycans Readily Ionize by Coordinating to Metal Ions, With Different Metal-adducts Enabling Isomeric Differentiation in Both Tandem MS and Ion Mobility-ms. However, Glycans Are Often Analyzed as Sodium Adducts Because So- Dium Is a Ubiquitous Contaminant. the Gallagher Lab Is Performing Systematic Studies Examining the Relation- Ship Between Metal-ion Properties and Glycan Characterization by MS. Finally, the Gallagher Lab Is Applying Native MS to Characterize Protein Complexes. in Native MS, Noncovalent Interactions Are Maintained in the Gas Phase; Yet Past Work Has Suggested That Proteins Analyzed as Positive Versus Negative Ions Have Differ- Ences in Gas-phase Stability. the Gallagher Lab Will Examine How Charging in Esi to Form Either Positive or Neg- Ative Ions Is Related to Gas-phase Protein Structure, Stability, and Ligand-binding Interactions. the Overarching Goal of the Gallagher Lab Is to Develop and Apply Novel Methodologies to Characterize Gly- Cans, Proteins, and Their Binding Interactions. the Research Described in This Proposal Is Significant Because It Will Provide a Fundamental Perspective on Esi-ms and the Molecular Insights From This Research Will Enable the Rational Development of Future Esi-ms Methods for Characterizing These Molecules. Ultimately, This Work Will Facilitate the Analysis of Glycans, Glycoconjugates, and Protein Complexes in Cell Biology and Disease States, Enabling These Methods to Be Applied to Address Important Biological Hypotheses.