BPS2026 – Mapping interactions of residual water in dry protein by solid-state X-ray footprinting mass spectrometry (ssXFMS)

As protein is lyophilized, the water content decreases to approximately 10% (w/w) of total mass. This retained water is difficult to disassociate from protein powder, requiring temperatures over 100°C to measure by thermogravimetric analysis, which suggests that the water is tightly bound to protein or excipients. Mapping protein-water interactions will complement other dry-state protein structure tools (such as LOVE NMR) and may help elucidate why some proteins rehydrate robustly, while other proteins cannot be rehydrated and appear to misfold irreversibly. To map protein-water interactions in dry protein, we have developed solid-state X-ray footprinting mass spectrometry (ssXFMS). In ssXFMS, dry protein is exposed to high power x-rays to generate hydroxyl radicals from residual water molecules. These radicals oxidize nearby side chains, leading to +16 Da modifications. To map these modifications, samples are rehydrated, proteolyzed, and analyzed by LC/MS. X-ray dose dependence of +16 Da modifications have been observed in multiple different lyophilized proteins, indicating that ssXFMS does indeed access protein-water interactions in dry powders. We are now investigating how these protein-water interactions change by addition of excipients or method of drying.