Quantitative proteomics analysis of adsorbed plasma proteins classifies nanoparticles with different surface properties and size

Abstract

Nanoparticle biological activity, biocompatibility and fate can be directly affected by layers of readily adsorbed host proteins in biofluids. Here, we report a study on the interactions between human blood plasma proteins and nanoparticles with a controlled systematic variation of properties using 18O-labeling and LC-MS-based quantitative proteomics. We developed a novel protocol to both simplify isolation of nanoparticle bound proteins and improve reproducibility. LC-MS analysis identified and quantified 88 human plasma proteins associated with polystyrene nanoparticles consisting of three different surface chemistries and two sizes, as well as, for four different exposure times (for a total of 24 different samples). Quantitative comparison of relative protein abundances was achieved by spiking an 18O-labeled “universal” reference into each individually processed unlabeled sample as an internal standard, enabling simultaneous application of both label-free and isotopic labeling quantification across the entire sample set. Clustering analysis of the quantitative proteomics data resulted in distinctive patterns that classified the nanoparticles based on their surface properties and size. In addition, temporal data indicated that the formation of the stable protein corona was at equilibrium within 5 min. The comprehensive quantitative proteomics results obtained in this study provide rich data for computational modeling and have potential implications towards predicting nanoparticle biocompatibility.

The Impact of Nanoparticle Protein Corona on Cytotoxicity, Immunotoxicity and Target Drug Delivery

“…For example, although blood plasma is constituted by thousands of proteins (≈4000) and some of which are more abundant than others [25]), their abundance in the plasma does not correspond to their abundance in the PC [26][27][28][29][30]. Moreover, the affinity-based competition between proteins for adsorption on the NP surface is responsible for the changes in the PC composition over time [31].…”

The on-bead digestion of protein corona on nanoparticles by trypsin immobilized on the magnetic nanoparticle

“…Rather than the isolation of protein corona from nanoparticles via SDS elution and the following precipitation with organic solvents, the direct digestion of protein corona on nanoparticles (also called on-bead digestion) might be an efficient approach to avoid the interference of SDS. With free trypsin, the protein corona on nanoparticles could be directly digested, which could simplify the procedures of the isolation and digestion of protein corona from nanoparticles [27]. However, the long time digestion of free trypsin is required to complete the digestion of proteins from nanoparticles for abovementioned on-bead digestion or SDS elution assay.…”

Biomolecular coronas provide the biological identity of nanosized materials

“…For example, as with any surface coverage phenomenon, the ratio of nanoparticle surface area to protein concentration is an important factor 1,14 . Still, there are strong indications that radius of curvature of the nanoparticle is also a key parameter for several reasons 1,5,13,18,19 .…”

“…Furthermore, these tens of proteins rarely correspond to the most abundant proteins in plasma, and are not necessarily those with the highest individual affinity to the nanoparticle surface 5,6,[18][19][20][21][22][23][24] .…”