Characterization of the structure of binary and ternary adsorbed protein films using electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectrometry, and radiolabeling

In complex adsorbed protein films, the biological reactivity of the adsorbed proteins depends on their relative concentrations, organization, conformation, and orientation. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) samples the outermost composition of an adsorbed protein film, providing biologically relevant information about the organization and availability of the adsorbed proteins in the film. We have previously shown that ToF-SIMS can quantitatively measure the composition of binary adsorbed protein films. However, for one particular binary system studied (immunoglobulin G-fibrinogen), a difference between the ToF-SIMS and I-125-radiolabeled protein adsorption measurements was reported. It was hypothesized that the composition of the protein film at its outermost (and, therefore, most biologically relevant) surface was different than that of the overall protein film due to the protrusion of the adsorbed fibrinogen over the adsorbed immunoglobulin G. This study provides further evidence supporting this hypothesis by incorporating electron spectroscopy for chemical analysis (ESCA) to examine the overall thickness of the adsorbed protein layers. Binary mixtures of three important plasma proteins (albumin, fibrinogen, and immunoglobulin G) and a ternary mixture of all three proteins were studied using radiolabeled protein adsorption measurements, ESCA, and ToF-SIMS. The different types of information generated using these techniques are shown to be complementary in describing the structure (i.e., organization, composition, conformation, and orientation) of the adsorbed protein films. Models of the proteins in the binary and ternary films are proposed based on the experimental data that reflect the apparent preferential location of fibrinogen at the surface for mixtures of fibrinogen and immunoglobulin G.