Investigation on topology-dependent adsorption and aggregation of protein on nanoparticle surface enabled by integrating time-limited proteolysis with cross-linking mass spectrometry

Abstract

The biological identity of nanomaterials is predominantly dictated by their surface protein corona (PC), yet the topological characteristics of most PCs remain uncharacterized in situ. We employed time-limited proteolysis combined time-segmented cross-linking mass spectrometry at specific intervals (10 min, 1 h, 2 h, 4 h and 18 h) to, for the first time, elucidate the spatial distribution, topological architecture and molecular orientation of multiple proteins within the multi-layered PC on nano-Fe₃O₄ surfaces. Additional monolinks, intermolecular and intramolecular crosslinks which were previously inaccessible to the crosslinker were unveiled in a layer-by-layer manner. 197 sparse intermolecular crosslinks involving 368 distinct wheat proteins were identified. Notably, charge complementarity and hydrophobic residue pairings, rather than hydrophobic peptide motifs, primarily govern the protein-protein interactions. For the crosslinks bridging the proteolysable and proteolysis-resistant layers, 72 % presented one end in a random coil conformation. Furthermore, the molecular orientation of 16 proteins including Q8L803, P11534 and P93594, etc., in the proteolysis-resistant layer was determined. The observation of violated intramolecular crosslinks between two rigid structural domains (e.g., A0A3B5Y430) suggests that nanoparticle-protein and protein-protein interactions may induce conformational changes in the adsorbed proteins. These findings offer novel insights into the spontaneous formation mechanisms of PC.

Keywords: Crosslink; Protein corona; Topological structure.