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Abstract Robust characterization of the protein corona—the layer of proteins that spontaneously forms on the surface of nanoparticles immersed in biological fluids—is vital for prediction of the safety, biodistribution, and diagnostic/therapeutic efficacy of nanomedicines. Protein corona identity and abundance characterization is entirely dependent on liquid chromatography coupled to mass spectroscopy (LC-MS/MS), though the variability of this technique for the purpose of protein corona characterization remains poorly understood. Here we investigate the variability of LC-MS/MS workflows in analysis of identical aliquots of protein coronas by sending them to different proteomics core-facilities and analyzing the retrieved datasets. While the shared data between the cores correlate well, there is considerable heterogeneity in the data retrieved from different cores. Specifically, out of 4022 identified unique proteins, only 73 (1.8%) are shared across the core facilities providing semiquantitative analysis. These findings suggest that protein corona datasets cannot be easily compared across independent studies and more broadly compromise the interpretation of protein corona research, with implications in biomarker discovery as well as the safety and efficacy of our nanoscale biotechnologies. 

Phase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well-established drivers of phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity. More specifically, phase separation of mixtures between RNA and positively charged proteins is described from a combination of multiscale computer simulations with microscopy and spectroscopy experiments. Phase diagrams were mapped out as a function of molecular concentrations in experiment and as a function of molecular size and temperature via simulations. The resulting condensates were found to retain at least some degree of internal dynamics varying as a function of the molecular composition. The results suggest a more general principle for phase separation that is based primarily on electrostatic complementarity without invoking polymer properties as in most previous studies. Simulation results furthermore suggest that such phase separation may occur widely in heterogenous cellular environment between nucleic acid and protein components.