Based on the investigation of fragment ions from proteins, information on the conformation and orientation of the proteins can be evaluated using TOF-SIMS spectra. TOF-SIMS produces important fragment ions with information regarding chemical structures at the uppermost surfaces of the samples. The surface is bombarded with a pulsed primary ion beam, inducing the desorption of particles. Because of the matrix effect, the desorption probability of particles and the formation probability of particular secondary ions strongly depend on the surface chemical structure, orientation, and molecular ambience (i. e., TOF-SIMS can be used to analyze not only the surface composition per se, but also the orientation and steric arrangement of molecules).
Since the sampling depth of TOF-SIMS in the static mode is approximately 1 nm, a thickness much less than for most proteins, TOF-SIMS is able to detect information about the conformation and orientation of proteins on substrates. There are two interesting approaches to the conformation and orientation of proteins on substrates that have been proposed in recent publications: one (Leufgen et al. 2003) is based on SF5 primary ions and the gentle ionization effect of gold substrate, and others (Coullerez et al. 2003; Xia and Castner 2003) are based on spectrum analysis by PCA.
Leufgan et al. (2003) have reported the investigation of a template-assembled synthetic (TASP) protein conformation and its orientation in self-assembled monolayers (SAM) by means of TOF-SIMS. In this study, both the presence and molecular orientation of the TASP molecule can be monitored by TOF-SIMS, and furthermore, the influence of self-assembly protocols on the TASP molecular surface orientation, which can be studied though Fourier transform infrared spectroscopy and surface plasmon resonance are not able to offer data applicable to these targets.
Xia et al. (Xia and Castner 2003; Xia et al. 2002) have investigated the preservation of the structure of adsorbed protein film during drying procedures in ultrahigh vacuum surface analysis techniques such as SIMS. Two methods, trehalose protection and glutaraldehyde fixation, were studied to preserve adsorbed protein structures under an ultrahigh vacuum conditions. Using the combination of PCA and static TOF-SIMS, it was found that trehalose protection could reduce the conformational change offibrinogen upon drying, and in particular prevent it from unfolding and thus exposing its hydrophobic domains. In addition, it has been indicated that the other method, glutaraldehyde fixation, is a valuable alternative stabilizing method, since the structural differences observed between glutaraldehyde-fixed protein films before and after drying are very similar to those observed between trehalose-protected and -unprotected dried protein films.
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