Time-of-flight secondary ion mass spectrometry (ToF-SIMS) characterization of conformation and orientation of adsorbed protein films.

摘要

This dissertation investigated the feasibility of static time-of-flight secondary ion mass spectrometry (ToF-SIMS) for analyzing the conformation and orientation of protein on surface.; One crucial step in this dissertation was to develop feasible preservation techniques for preparing adsorbed protein samples for ToF-SIMS analysis. This was because proteins tend to change its conformation in the absence of water. One promising preservation method identified in this dissertation was trehalose protection. Surface plasmon resonance (SPR) measurements showed that trehalose-protected dried antibodies films retained a much higher proportion of their hydrated antigen binding activity than the adsorbed protein films without trehalose protection. By applying principal component analysis (PCA) to the ToF-SIMS spectra of the adsorbed protein films, the biological activity difference observed in SPR was correlated to changes in protein conformation. Trehalose protected proteins retained a greater degree of their original conformation than the unprotected proteins. The other preservation method, glutaraldehyde fixation was also examined in this dissertation. It was found the structural differences observed between protein films glutaraldehyde fixed before drying and after drying have similarity to those observed between trehalose protected dried fibrinogen films and unprotected dried fibrinogen films, thus both preservation methods could prevent the protein from unfolding and exposing hydrophobic domains.; Also in this dissertation, a model system was developed for examining the feasibility of ToF-SIMS to characterize the orientation of proteins on surface. In the reaction scheme, nitrilotriacetic acid (NTA) was immobilized onto a self-assembled monolayer (SAM) of oligo(ethylene glycol)-terminated (OEG) thiol on gold. SPR analysis confirmed that after nickel activation, the NTA/OEG surface could specifically bind his-tagged protein. Moreover, an immobilized his-tagged antibody fragment (HuLys Fv) had nearly full antigen (lysozyme) binding capacity, suggesting a uniform complimentarity determining region (CDR)-exposed orientation of HuLys Fv on the surface. For comparison, the OEG SAM was also activated with carbonyldiimidazole (CDI). Proteins should bind the CDI-activated surface with random orientation. This was supported by SPR results, which showed that only ∼50% of HuLys Fv immobilized on the CDI/OEG surface had antigen binding capability.