Influence of the dispersion medium, washing medium, and digestion method on the composition of the hard protein corona of nanoparticles

Protein corona analysis of nanoparticles involves multiple steps, each of which has a huge impact on the final composition of the protein corona. In this study we focus on the impact of chosen dispersion media, washing media, and digestion methods. Therefore, nanoparticles were fabricated and subsequently dispersed in either water or phosphate-buffered saline (PBS) and afterwards incubated with human plasma. To remove excess proteins, the samples were centrifuged and then washed in water or PBS. Four different digestion methods were tested, to prepare the samples for protein identification and quantification by mass spectrometry analysis. Our results showed that fewer proteins with a high isoelectric point and less immunoglobulins were identified when using PBS as dispersion and washing medium instead of water. Especially for quantification of proteins in the protein corona, the choice of dispersion and washing media had a huge impact on the results and PBS should preferably be used. Of the tested digestion methods, the filter-aided sample preparation method leads to the most identified proteins, and the highest label-free quantification intensities could be measured, making it the method of choice. However, an in-solution method and two different on-particle digestion methods also showed promising numbers of identified and quantified proteins, so they are less labor-intensive alternatives to the filter-aided sample preparation method.