Quantitative cross-linking mass spectrometry for protein structural stability studies

Abstract

Mass spectrometry-based methods for protein-ligand identification have expanded classical techniques for the bioanalytical characterization of small molecule target engagement and their modes of action. In the last decade, a series of techniques have coupled mass spectrometry readout, structure-function framework, and thermodynamic stability to expand the suite of proteomics techniques for protein-ligand interactions. Although these methods have proven powerful, due to the complex nature of these large-scale studies, having multiple avenues of assessment is critical for the proper evaluation of clinical value. In this work, the interfacing of these protein-denaturation experimental designs with cross-linking mass spectrometry sample workflows is investigated to better understand the protein topologies in these protein-ligand large-scale analyses. The developed method, protein-denaturation and quantitative cross-linking mass spectrometry, offers another strategy in the unbiased assessment of protein target engagement studies. Additionally, from a basic science perspective, this method also provides data in understanding the molecular principles of protein folding in structure-(dys)function studies. First, I validated a proof-of-concept of protein-denaturation with quantitative cross-linking mass spectrometry in a standard protein and known ligand. Then, I adapted and assessed the viability of this method on the proteome-level scale. Although this method has much room for optimization for tackling large-scale studies, its data provides promise with smaller complex proteomes. Overall, quantitative cross-linking mass spectrometry during protein unfolding is a reliable assay that can be used alone or provide complementary information to the current generation of protein-denaturation mass-spectrometry methods for generating target-engagement atlases.