Evaluation of desorption of proteins adsorbed to hydrophilic surfaces by two-dimensional electrophoresis.
The evaluation of the plasma protein adsorption patterns of superparamagnetic iron oxide (SPIO) particles is of high interest concerning their in vivo fate and is carried out by two-dimensional electrophoresis (2-DE). The sample preparation is of great importance, especially the removal of the adsorbed proteins (desorption) from the particle surface for subsequent analysis by 2-DE. The removal is carried out by a desorption solution. In this study, negatively and positively charged SPIO model particles were under investigation concerning the desorption of proteins adsorbed on their surfaces. Firstly, the desorption process was determined quantitatively using the Bradford protein assay. Secondly, the removable or nonremovable protein species, from particles surface were under investigation by 2-DE. Looking at the desorption in a quantitative manner with the Bradford assay, the desorption efficacy from negatively charged particles was about 90%. In the case of the positively charged particles, the desorption efficacy seemed to be reduced, approximately 34% of the proteins remained on the surface. Comparing the protein patterns of the particles evaluated by 2-DE in the desorption solution and the proteins remaining on the particles, they confirmed the results 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 species were found after desorption. The more basic the protein fragments, the more ineffective was the desorption from the positively charged model particle, and vice versa. Nevertheless, all protein spots were found qualitatively in the desorption solution, especially when the desorption solutions still containing the particles were used for the 2-DE analysis. In conclusion, 2-DE could be confirmed as the "gold standard" for determining the plasma protein adsorption patterns of nanoparticulate systems.
Lindl
K
K
Department of Pharmaceutical Technology, The Free University of Berlin, Germany.
Kresse
M
M
Müller
R H
RH
D000042:Absorption; D001681:Biological Assay; D001797:Blood Protein Electrophoresis / Q000379:methods*; D001798:Blood Proteins / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005290:Ferric Compounds / Q000737:chemistry; D005782:Gels / Q000737:chemistry; D006801:Humans
D001798:Blood Proteins; D005290:Ferric Compounds; D005782:Gels; C000499:ferric oxide