Abstract:
The vast majority of biological processes such as cell-cell communication, immune response, reaction catalysis and the protein transport are driven by protein-protein interactions (PPIs). Elucidation of PPIs, which when altered are associated with various pathophysiological processes, opened an emerging era of protein analysis. The knowledge of the PPIs interfaces is crucial to obtain deeper insights into the PPIs mode of action and how to modulate them. In particular, PPIs between antibodies and antigens are of great interest for diagnostic and therapeutic applications in the biopharmaceutical industry and scientific research. In addition to high-resolution techniques such as nuclear magnetic resonance spectroscopy (NMR), X-ray crystallography and cryo-electron microscopy (cryo-EM), hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) has evolved to a powerful analysis method for the elucidation of PPI interfaces. Without introducing structural modifications, HDX-MS can be used to elucidate protein dynamics and protein-ligand interaction sites of proteins of almost unlimited size, with high tolerance against impurities, low sample consumption and reasonable resolution and throughput. In addition, HDX data reflect the native in-solution protein conformation. However, HDX-MS remains challenging for the analysis of proteins encompassing multiple post-translational modifications such as disulphide bonds and N-glycosylations.
In the context of the present work, a setup and workflow for HDX-MS bottom-up analyses was established. A prerequisite for reliable and efficient HDX analyses is the precise control of the pH, temperature and timing, which was facilitated by a tailor made Semi-Automated Interface for HD exchange (SAIDE). The SAIDE enables flexible use of a HPLC-MS instrumentation for various analyses, efficient use of the laboratory space while displaying a cost efficient approach compared to fully automated, commercial HDX systems.
With this setup, a protocol for HDX analyses was developed and optimised, which addressed critical generic HDX parameters such as efficient proteolysis and good reproducibility while keeping the back exchange as low as possible. Compared to dissolved pepsin, the proteolysis efficiency could be increased using pepsin immobilized on beads. This enabled a tenfold reduction in digestion time while achieving a higher number of peptides, run-to-run recovery, a lower HDX variance, and lower average peptide length. These optimisations were performed as part of an HDX study designed to elucidate the binding region of a clinical mAb drug candidate targeting Annexin-A1 in a calcium dependent manner.
Subsequently, the HDX-MS protocol was improved in terms of sample throughput and adapted to another protein target, the receptor-binding domain (RBD) of SARS-CoV-2. The high reproducibility of the established protocol enabled its application in an HDX screening workflow using a lower number of deuteration time points. Epitopes of seven nanobodies were characterized within a time period of roughly four weeks. The HDX-MS screening approach was used to support evidence based identification of two lead candidates potent in their viral neutralization.
Subsequent endeavours aimed to adapt the established screening approach to proteins encompassing multiple post-translational modification (PTMs). While extracellular proteins display attractive drug targets or are themselves used as biopharmaceuticals, nearly each of them encompass multiple PTMs such as N-glycosylated and disulphide bonds. Originated in their heterogeneity, N-glycosylations remain challenging for structural analysis such as HDX-MS. Here, a novel peptide N-glycanase from Rudaea cellulosilytica (Rc) was characterized that exhibits broad substrate specificity and high activity for deglycosylation of natively folded proteins. Thus, the enzyme was used to facilitate MS based top-down protein analytics and offers the opportunity for N-deglycosylation of peptides in several minutes. Moreover, following heterologous expression the enzyme can be obtained from E. coli with high yield sufficient purity. Due to its acidic pH optimum, the PNGase Rc was successfully used under challenging HDX-MS quenching conditions (0 °C; pH 2.5) in presence of commonly applied concentrations of reducing and denaturing agents Tris(2-carboxyethyl)phosphine (TCEP), urea and guanidinium chloride (GdmCl). As a proof-of-principle the PNGase Rc was integrated into the established HDX epitope screening workflow (post-proteolysis) resulting in the elucidation of four nanobody epitopes targeting the multiple N-glycosylated extracellular domain of the signal-regulatory protein alpha (SIRPα). The additional deglycosylation increased sequence coverage and redundancy and also enabled the detection of epitopes in proximity of N-glycosylation sites.
Mass Spectrometry
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