Mapping ligand-protein interactions through structural proteomics and mass spectrometry

<jats:p>&lt;p dir="ltr"&gt;Proteins are the primary targets of most therapeutic drugs, and understanding how small molecules interact with proteins is fundamental to drug discovery and development. While numerous approaches have been developed to identify drug targets, many remain limited in their ability to localize ligand-binding sites, achieve high throughput, or comprehensively characterize structural changes in proteins. Advances in structural proteomics have created new opportunities to investigate protein-ligand interactions directly in complex biological systems.&lt;/p&gt;&lt;p dir="ltr"&gt;The overall aim of this thesis was to develop and apply mass spectrometry- based structural proteomics methods for mapping ligand-protein interactions and characterizing protein structural changes. Particular focus was placed on exploiting limited proteolysis to improve drug target identification, binding-site localization, and proteome-wide structural analysis.&lt;/p&gt;&lt;p dir="ltr"&gt;In Paper I, Above-Filter Digestion Proteomics (AFDIP) was introduced as a method for identifying drug targets while simultaneously localizing ligand- binding regions through differential protease accessibility. The approach was validated using several model compounds and demonstrated broad applicability for studying protein-ligand interactions. In Paper II, the cleavage specificity of trypsin in native proteins was systematically investigated, providing insights into the structural determinants of proteolysis and establishing a foundation for interpreting limited proteolysis experiments. Paper III described Auto-AFDIP, an automated implementation of AFDIP that substantially increased throughput, reproducibility, and scalability while maintaining analytical performance. Finally, Paper IV presented HigH-ratiO partial proteolysis with carriER proteome (HOLSER), a highly sensitive strategy that combines partial proteolysis with carrier proteomes to enable proteome-wide structural profiling and site- resolved mapping of ligand-binding events.&lt;/p&gt;&lt;p dir="ltr"&gt;Together, the work presented in this thesis expands the structural proteomics toolbox by introducing complementary methodologies for investigating protein structure and ligand interactions. These approaches improve the sensitivity, throughput, and structural resolution of mass spectrometry-based analyses and provide new opportunities for studying protein function, drug mechanisms of action, and the development of safer and more effective therapeutics.&lt;/p&gt;&lt;h3 dir="ltr"&gt;List of scientific papers&lt;/h3&gt;&lt;p dir="ltr"&gt;I. &lt;b&gt;Sokolova B,&lt;/b&gt; Gharibi H, Jafari M, Lyu H, Lovera S, Gaetani M, Saei AA, Zubarev RA. Above-Filter Digestion Proteomics Reveals Drug Targets and Localizes Ligand Binding Site. Journal of Proteome Research. 2026; 25:1556-1570. &lt;a href="https://doi.org/10.1021/acs.jproteome.5c00927" target="_blank" rel="noreferrer"&gt;https://doi.org/10.1021/acs.jproteome.5c00927&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;II. Gaspar M, &lt;b&gt;Sokolova B,&lt;/b&gt; Saei AA, Marques JC, Zubarev RA. Beyond the Known Cuts: Trypsin Specificity in Native Proteins. Chemical Communications. 2025; 61:12753-12756. &lt;a href="https://doi.org/10.1039/d5cc02378e" target="_blank" rel="noreferrer"&gt;https://doi.org/10.1039/d5cc02378e&lt;/a&gt;&lt;/p&gt;&lt;p dir="ltr"&gt;III. &lt;b&gt;Sokolova B+&lt;/b&gt;, Meng Z+, Gaetani M, Zubarev RA. Automated Above-Filter Digestion Proteomics for High-Throughput Drug Target Identification. +These authors contributed equally to this work. [Manuscript]&lt;/p&gt;&lt;p dir="ltr"&gt;IV. Zhang X, &lt;b&gt;Sokolova B,&lt;/b&gt; Meng Z, Gharibi H, Lyu H, Saei AA, Gaetani M, Zubarev RA. HigH-ratiO partial proteolysis with carriER proteome (HOLSER) Enables Global Structure Profiling and Site-resolved Elucidation of Ligand-Protein Interactions. bioRxiv. 2025. [Manuscript Preprint] &lt;a href="https://doi.org/10.1101/2025.07.11.664381"&gt;https://doi.org/10.1101/2025.07.11.664381&lt;/a&gt;&lt;br&gt;&lt;/p&gt;</jats:p>