Connecting charge, structure, and biomolecular interactions with native mass spectrometry
Authors: Abramsson Mia 
Published: January 2025

<p dir="ltr">Understanding biomolecular interactions is crucial for advancing our fundamental knowledge of biological processes and for aiding pharmaceutical development to target diseases effectively. This thesis aims to investigate how protein properties and biomolecular interactions influence the charging mechanisms during electrospray ionization (ESI) in native mass spectrometry (nMS). We then use the insigths from these studies to investigate the structure-function relationship of membrane protein-lipid complexes.</p><p dir="ltr">Paper I examines the role of ionizable residues in ESI charging using protein engineering across three different systems. We find that in the absence of ionizable residues, proteins can still charge according to their surface area, but exhibit a lower proton affinity and gas-phase stability. The position of these residues also affects protein stability due to Coulombic destabilization.</p><p dir="ltr">Paper II rigorously investigates how the choice of detergents impacts the final charge state of membrane proteins. The study highlights the role of the detergent headgroup's proton affinity in membrane protein charging and suggests a charge equilibration mechanism during detergent removal in the gas-phase. This equilibration occurred through the transfer of protons from the detergent headgroup to the transmembrane region of the protein in the vacuum environment. This process thus be modulated to stabilize labile protein complexes for nMS analysis.</p><p dir="ltr">Paper III explores the structure-charge state relationships of protein-DNA complexes reported in the literature. While DNA complexes exhibit slightly lower charges compared to proteins, they both follow a power-law relationship. The charge state distributions vary significantly for protein-DNA complexes. We observe that the final charge state differs depending on whether the DNA is bound centrally or peripherally, which can be explained by the higher density and gas- phase compaction of DNA molecules.</p><p dir="ltr">Paper IV investigates the influence of low-complexity domains on protein ionization. Using both designed and naturally occurring proteins containing a mixture of folded and low-complexity domains, the study elucidates their roles in the charging mechanism, facilitating better interpretation of these processes.</p><p dir="ltr">Paper V focuses on an artificial membrane protein system to identify the fundamental principles of lipid binding and lipid-mediated stabilization. By combining nMS with molecular dynamics simulations, the study uncovers key features, including residue positioning and dynamics involved in lipid-mediated stabilization. Additionally, it led to the discovery of a novel cardiolipin binding site in a membrane-bound protease, which plays a role in the protein's allosteric regulation.</p><p dir="ltr">Togheter, the findings from these five studies shed light on the fundamental mechanisms of ESI and demonstrate how protein engineering, alongside biomolecular interactions, can be leveraged to gain deeper insights into protein structure and function.</p><h3>List of scientific papers</h3><p dir="ltr">I. MIA L. ABRAMSSON, Cagla Sahin, Jonathan T. S. Hopper, Rui M. M. Branca, Jens Danielsson, Mingming Xu, Shane A. Chandler, Nicklas Österlund, Leopold L. Ilag, Axel Leppert, Joana Costeira-Paulo, Lisa Lang, Kaare Teilum, Arthur Laganowsky, Justin L. P. Benesch, Mikael Oliveberg, Carol V. Robinson, Erik G. Marklund, Timothy M. Allison, Jakob R. Winther & Michael Landreh. Charge engineering reveals the roles of ionizable side chains in electrospray ionization mass spectrometry. JACSAu. 2021, 1, 2385-2393. <a href="https://pubs.acs.org/doi/10.1021/jacsau.1c00458" rel="noreferrer" target="_blank">https://pubs.acs.org/doi/10.1021/jacsau.1c00458</a></p><p dir="ltr">II. Hsin-Yung Yen, MIA L. ABRAMSSON, Mark T. Agasid, Dilraj Lama, Joseph Gault, Idlir Liko, Margit Kaldmäe, Mihkel Saluri, Abdul Aziz Qureshi, Albert Suades, David Drew, Matteo T. Degiacomi, Erik G. Marklund, Timothy M. Allison, Carol V. Robinson & Michael Landreh. Electrospray ionization of native membrane proteins proceeds via a charge equilibration step. RSC Advances. 2022, 12, 9671-9680. <a href="https://doi.org/10.1039/d2ra01282k" rel="noreferrer" target="_blank">https://doi.org/10.1039/d2ra01282k</a></p><p dir="ltr">III. MIA L. ABRAMSSON, Louise J. Persson, Frank Sobott, Erik G. Marklund & Michael Landreh. Charging of DNA Complexes in Positive-Mode Native Electrospray Ionization Mass Spectrometry. J. Am. Soc. Mass. Spectrom. 2024, 35, 12, 3157-3162. <a href="https://doi.org/10.1021/jasms.4c00335" rel="noreferrer" target="_blank">https://doi.org/10.1021/jasms.4c00335</a></p><p dir="ltr">IV. Hannah Osterholz, Alexander Stevens, MIA L. ABRAMSSON, Dilraj Lama, Anna Rising, Erik G Marklund, Arne Elofsson, Sebastian Deindl, Axel Leppert & Michael Landreh. Native mass spectrometry captures the conformational plasticity of proteins with low-complexity domains. JACSAu. 2025. <a href="https://doi.org/10.1021/jacsau.4c00961">https://doi.org/10.1021/jacsau.4c00961<br></a><br></p><p dir="ltr">V. MIA L. ABRAMSSON, Robin A. Corey, Jan Škerle, Louise J. Persson, Olivia Anden, Abraham O. Oluwole, Rebecca J. Howard, Erik Lindahl, Carol V. Robinson, Kvido Strisovsky, Erik G. Marklund, David Drew, Phillip J. Stansfeld & Michael Landreh. Engineering cardiolipin binding to an artificial membrane protein reveals determinants for lipid-mediated stabilization. eLife [Manuscript Preprint] <a href="https://doi.org/10.7554/eLife.104237.1" rel="noreferrer" target="_blank">https://doi.org/10.7554/eLife.104237.1</a></p>