How to effectively prepare a sample for bottom-up proteomic analysis of nanoparticle protein corona? A critical review

Since the interest in the biomedical applications of inorganic nanoparticles (NPs) has rapidly grown over the last decades, there is a need for a thorough characterization of bio-nano interactions. NPs introduced to the body (mostly intravenously) encounter plasma proteins, that instantly create a so-called protein corona on the NPs surface, giving the nanomaterial a new biological identity. Type of the proteins that interact with NPs may affect the in vivo fate of NPs. For that reason, it is particularly important to establish analytical methods capable of corona protein identification. Bottom-up proteomics is most often used for that purpose. A crucial part of the experiment is sample preparation, as it is already proven that different protocols may lead to distinct results. This review is aimed at providing a characterization of two main stages of sample preparation: separation of NPs with protein corona from the unbound proteins and the digestion of corona proteins. Separation techniques such as centrifugation, magnetic separation, and chromatography and three digestion methods (in-gel, in-solution, and on-particle) are described with special emphasis paid on their advantages and disadvantages as well as their influence on the result of identification. This paper also indicates the need for standardization of protein corona identification protocols, as some of the proteins may be preferentially detected while applying a particular digestion procedure.

Automated summary

With the growing interest in the biomedical applications of inorganic nanoparticles (NPs) in recent decades, there is a need for detailed characterization of bio-nano interactions, particularly the protein corona formed on NP surfaces. Analytical methods capable of identifying corona proteins are essential, and bottom-up proteomics is commonly used for this purpose. The experiment's sample preparation, including separation techniques and digestion methods, plays a crucial role in the identification process, with distinct protocols leading to different outcomes. Standardization of protein corona identification protocols is needed to prevent preferential detection of certain proteins with specific digestion procedures.