Quantitative analysis of binary adsorbed protein films by time of flight secondary ion mass spectrometry

Time of flight secondary ion mass spectrometry (ToF-SIMS) is an ideal technique for the analysis of adsorbed protein films because of its surface sensitivity and chemical specificity. In this study, we examined ToF-SIMS with the multivariate calibration method partial least squares regression (PLSR) for the determination of the relative abundance of the components in binary protein films adsorbed onto mica, PTFE, and heptyl amine plasma polymer substrates. These results have been compared with independently measured 125I-radiolabeled protein adsorption experiments. By applying PLSR to the ToF-SIMS data, the relative abundance of the components in the binary adsorbed protein films was quantified, and the agreement between the ToF-SIMS and 125I-radiolabeling data was measured by the root mean square prediction error (RMSPE). Differences in protein quantification by PLSR and 125I-radiolabeling ranged from 5 to 25 mass % RMSPE and were highly dependent on the structure of the adsorbed protein film, the substrate surface chemistry and morphology, and the number of latent variables retained in the PLSR model. The limit of detection for the minor component in the adsorbed protein film was found to be approximately 10 mass %. This study demonstrates that the combination of ToF-SIMS and multivariate calibration provide complementary information to 125I-radiolabeling about the composition and structure of binary adsorbed protein films.