Evaluation of desorption of proteins adsorbed to hydrophilic surfaces by two-dimensional electrophoresis

The evaluation of the plasma protein adsorption patterns of superparamagnet ic iron oxide (SPIO) particles is of high interest concerning their in vivo fate and is carried out by two-dimensional electrophoresis (2-DE). The sam ple preparation is of great importance, especially the removal of the adsor bed proteins (desorption) from the particle surface for subsequent analysis by 2-DE. The removal is carried out by a desorption solution. In this stud y, negatively and positively charged SPIO model particles were under invest igation concerning the desorption of proteins adsorbed on their surfaces. F irstly, the desorption process was determined quantitatively using the Brad ford protein assay. Secondly, the removable or nonremovable protein species , from particles surface were under investigation by 2-DE. Looking at the d esorption in a quantitative manner with the Bradford assay, the desorption efficacy from negatively charged particles was about 90%. In the case of th e positively charged particles, the desorption efficacy seemed to be reduce d, approximately 34% of the proteins remained on the surface. Comparing the protein patterns of the particles evaluated by 2-DE in the desorption solu tion and the proteins remaining on the particles, they confirmed the result s from the protein quantification. After desorption, the IgG gamma -chains were found to be the dominant protein fraction remaining on the negatively charged particles. On the positively charged particles, many more protein s pecies were found after desorption. The more basic the protein fragments, t he more ineffective was the desorption from the positively charged model pa rticle, and vice versa. Nevertheless, all protein spots were found qualitat ively in the desorption solution, especially when the desorption solutions still containing the particles were used for the 2-DE analysis. In conclusi on, 2-DE could be confirmed as the "gold standard" for determining the plas ma protein adsorption patterns of nanoparticulate systems.