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Quantitative profiling of the protein coronas that form around nanoparticles

Nature Protocols volume 9, pages 2030–2044 (2014)Cite this article

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Abstract

Nanoparticle applications in biotechnology and biomedicine are steadily increasing. In biological fluids, proteins bind to nanoparticles that form the protein corona, crucially affecting the nanoparticles' biological identity. As the corona affects in vitro and/or in vivo nanoparticle applications, we developed a method to obtain time-resolved protein corona profiles formed on various nanoparticles. After incubation in plasma or a similar biofluid, or after injection into a mouse, the first analytical step is sedimentation of the nanoparticle-protein complexes through a sucrose cushion, thereby allowing analysis of early corona formation time points. Next, corona profiles are visualized by gel electrophoresis and quantitatively analyzed after tryptic digestion using label-free liquid chromatography–high-resolution mass spectrometry. In contrast to other approaches, our established methodology allows the researcher to obtain qualitative and quantitative high-resolution corona signatures. The protocol can be readily extended to the investigation of protein coronas from various nanomaterials (as an example, we applied this protocol to different silica nanoparticles (SiNPs) and polystyrene nanoparticles (PSNPs)). Depending on the number of samples, the protocol from nanoparticle-protein complex recovery to data evaluation takes ∼8–12 d to complete.

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Figure 1
Figure 2: Schematic overview of the protocol's entire workflow.
Figure 3: Effect of residual blood plasma on corona profiles.
Figure 4: NanoUPLC setup for direct injection.
Figure 5: 1D gel electrophoresis to visualize nanoparticle-bound human plasma proteins.
Figure 6: Effect of different nanoparticle properties on corona profiles.

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Acknowledgements

Grant support for this study: Deutsche Forschungsgemeinschaft (DFG)-SPP1313, DFG-SFB490/Z3; Bundesministerium für Bildung und Forschung (BMBF)-MRCyte/NanoBEL/DENANA; Zeiss-ChemBioMed; University Mainz Forschungszentrum Immunologie; Research Center for Immunology (FZI); and Stiftung Rheinland-Pfalz (NANOSCH, NanoScreen).

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Author notes

  1. Dominic Docter and Ute Distler: These authors contributed equally to this work.

Authors and Affiliations

  1. Molecular and Cellular Oncology, ENT, University Medical Center of Mainz, Mainz, Germany

    Dominic Docter, Desirée Wünsch, Angelina Hahlbrock & Roland H Stauber

  2. Institute for Immunology, University Medical Center of Mainz, Mainz, Germany

    Ute Distler, Wiebke Storck, Jörg Kuharev & Stefan Tenzer

  3. Institute for Molecular Biology, University Duisburg-Essen, Essen, Germany

    Shirley K Knauer

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  1. Dominic Docter
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Contributions

S.T., R.H.S., J.K., U.D. and D.D. developed the protocol; D.D., U.D., J.K., A.H., W.S., D.W., S.K.K., S.T. and R.H.S. conducted the experiments, interpreted the data and drafted the manuscript.

Corresponding authors

Correspondence to Stefan Tenzer or Roland H Stauber.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Settings for ISOQuant post-processing for label-free quantification. (XLSX 11 kb)

Supplementary Table 2

Integrated summary of corona proteins identified on silica and polystyrene nanoparticles8,12, containing averaged (typical) abundance values (expressed in parts per million of total corona protein) for each nanoparticle type, including human plasma as a reference for future studies. The table also contains information regarding functional annotation, molecular weight and isoelectric points for corona-associated proteins. (XLSX 63 kb)

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Docter, D., Distler, U., Storck, W. et al. Quantitative profiling of the protein coronas that form around nanoparticles. Nat Protoc 9, 2030–2044 (2014). https://doi.org/10.1038/nprot.2014.139

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