Preserving the structure of adsorbed protein films for time-of-flight secondary ion mass spectrometry analysis.
The characterization of adsorbed protein films with ultrahigh vacuum (UHV) surface analysis techniques requires dehydration of the samples, which can cause significant alterations in protein structure. It is desirable to preserve the structure of adsorbed protein films during drying, so UHV analysis could be done in a state that is more representative of proteins' actual structure in the aqueous environment. In this study, two methods, trehalose protection and glutaraldehyde fixation, were explored for their feasibility in preserving adsorbed protein structure for a powerful UHV surface analysis technique, time-of-flight secondary ion mass spectrometry (ToF-SIMS). Trehalose protection had shown some promise for ToF-SIMS analysis in our previous study and was further examined with the model protein fibrinogen in this study. Using the combination of principal component analysis (PCA) and static ToF-SIMS analysis, we found that trehalose protection could reduce the conformation change of fibrinogen upon drying, and prevent it from unfolding and exposing hydrophobic domains. Moreover, when the adsorbed protein film became more densely packed, the drying-induced changes in protein structure were reduced. Thus, the protection afforded by trehalose coating was more significant at lower protein surface concentrations. The other method, glutaraldehyde fixation, was used in ToF-SIMS analysis for the first time. The epsilon-amino group of lysine was identified as the major reactive group in the protein structure toward glutaraldehyde fixation. Structural differences observed between fibrinogen films that were glutaraldehyde fixed before drying and after drying were similar to those observed between trehalose-protected and-unprotected dried fibrinogen films. Glutaraldehyde fixation was found to be a viable, alternative stabilizing method to trehalose protection for ToF-SIMS analysis.
D000327:Adsorption; D003432:Cross-Linking Reagents; D005340:Fibrinogen / Q000737:chemistry; D005976:Glutaral; D011506:Proteins / Q000737:chemistry*; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*; D014199:Trehalose
D003432:Cross-Linking Reagents; D011506:Proteins; D005340:Fibrinogen; D014199:Trehalose; D005976:Glutaral