Proteins sit at the very heart of life’s machinery, orchestrating almost every biological function necessary for cells and organisms to thrive. The concept of proteins, first coined by Swedish chemist Jöns Jacob Berzelius in the early 19th century, derived from the Greek proteios, meaning “primary” or “of first importance,” aptly highlights their foundational role. Despite the rudimentary understanding of their nature in those early days, it was undeniable that proteins were indispensable to life. Today, we comprehend that proteins often execute their roles through complex interactions with smaller molecules called ligands, which bind to specific sites on protein structures, influencing both their function and stability.
A novel technological breakthrough presented by researchers at the European Molecular Biology Laboratory (EMBL) now promises to revolutionize how scientists decode these protein-ligand interactions on a massive scale. In a recent study published in Nature Structural and Molecular Biology, the Savitski Group at EMBL unveiled HT-PELSA, a high-throughput peptide-centric local stability assay. This method not only scales up the classical PELSA workflow but also enhances sensitivity and applicability, allowing the exploration of protein-ligand engagements directly in complex biological mixtures such as crude cell lysates, tissues, and bacteria—a feat previously unattainable with traditional techniques.
The original PELSA methodology emerged only last year as an innovative technique to detect protein-ligand binding events by monitoring local changes in protein stability upon ligand association. Binding typically stabilizes specific protein regions against enzymatic cleavage by proteases such as trypsin, resulting in changes in the abundance of peptide fragments from those regions. Tracking such subtle alterations across the entire proteome allowed researchers to identify previously elusive binding hotspots with remarkable peptide-level resolution. However, PELSA’s labor-intensive nature restricted it to processing only a limited number of samples daily, imposing significant bottlenecks on throughput and sample diversity.
HT-PELSA brilliantly addresses these limitations through a radical shift in sample handling format, transitioning from traditional tubes to a 96-well plate micro-well system optimized for automation. This transformation enables robotic handling and parallel processing of hundreds of samples simultaneously, exponentially boosting throughput without compromising the exquisite sensitivity that made PELSA powerful. As Kejia Li, the pioneering postdoctoral fellow who spearheaded the adaptation at EMBL, explains, this newly developed workflow now allows the analysis of roughly 400 samples per day, compared to a mere 30 with the original method—a remarkable fifteenfold increase in efficiency.
At the core of HT-PELSA’s enhanced workflow lies an ingenious exploitation of the hydrophobic and water-repellant characteristics of proteins relative to their peptide fragments. While trypsin digestion retains its fundamental role, HT-PELSA leverages a novel protein-adsorption surface, which preferentially captures intact proteins while allowing cleaved peptides to remain in solution. This physical separation streamlines sample processing and, crucially, opens the door to investigating membrane proteins—a notoriously difficult class to analyze. Membrane proteins comprise about 60% of all known drug targets and their delicate structures often render them incompatible with conventional methods requiring harsh extraction and purification, which can alter their native conformations.
By enabling the direct study of protein-ligand binding events in unpurified complex biological samples, HT-PELSA offers unprecedented insights into physiologically relevant interactions. This method preserves the native environment of membrane proteins and other challenging targets, allowing researchers to observe how candidate drugs or endogenous molecules engage these proteins under biologically realistic conditions. Such capability is vital for drug discovery pipelines, where understanding the binding specificity and mechanistic impact of small molecules on their intended targets can dramatically improve target validation and lead optimization.
Isabelle Becher, a key contributor to the project and laboratory officer in charge at EMBL’s Savitski Group, emphasizes the profound biological understanding HT-PELSA provides. The method charts an expansive landscape of protein-ligand interactions by revealing dynamic changes in local stability patterns across thousands of proteins simultaneously. This holistic view enhances the ability to decipher underlying molecular mechanisms governing cellular processes and pathologies. Furthermore, identifying selective interactions aids in the rational design of therapeutics tailored to precise protein targets, improving efficacy while minimizing off-target effects, thus advancing safer and more effective medicines.
In addition to its primary focus on ligand binding, the current study showcases HT-PELSA’s capacity to detect modifications in protein-protein interactions induced by ligand association. This dual-sensitivity highlights the method’s versatility in capturing the wider network of molecular interactions that define cellular states and responses. Looking forward, the researchers intend to extend HT-PELSA’s application to assess protein-nucleic acid interactions, further expanding its utility in elucidating the molecular architecture and functional circuitry of cells.
Mikhail Savitski, team leader at EMBL Heidelberg and senior author of the study, remarks that HT-PELSA is a transformative step in proteomics technology, significantly accelerating both fundamental research and applied biomedical science. Beyond its immediate role in protein function characterization, HT-PELSA’s scalability and robustness position it as a cornerstone platform capable of driving large-scale drug screening campaigns. Its integration with advanced mass spectrometry and bioinformatics workflows promises to usher in a new era of molecular precision medicine.
This advancement holds particular promise for the pharmaceutical industry and academic researchers alike. The ability to swiftly assess hundreds of drug candidates across diverse protein targets, including integral membrane components often overlooked previously, represents a monumental stride toward combating complex diseases. Simultaneously, it equips biologists with a powerful tool to dissect proteome dynamics in various tissues and organisms, facilitating a deeper grasp of biology from a systems-level perspective.
In summary, HT-PELSA marks a milestone in the ongoing quest to decode the intricacies of protein-ligand interactions at an unprecedented scale and depth. By marrying the finesse of the original PELSA assay with automation and novel biochemical innovations, this cutting-edge technology unlocks access to challenging protein classes and complex biological samples. It stands as a shining example of how marrying technological ingenuity with biological insight can create transformative tools, primed to accelerate the discovery and optimization of next-generation drugs while enriching our fundamental understanding of life’s molecular underpinnings.
Subject of Research: Protein-ligand interactions; high-throughput proteomics; membrane proteins; drug discovery
Article Title: High-throughput peptide-centric local stability assay extends protein–ligand identification to membrane proteins, tissues and bacteria
News Publication Date: 5-Nov-2025
Web References: 10.1038/s41594-025-01699-y
Image Credits: Daniela Velasco/EMBL
Keywords: Molecular evolution, Proteomics
