Quantitative profiling of the protein coronas that form around nanoparticles

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via

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NANOPARTICLE PROTEIN CORONA ACROSS CORE FACILITIES Article Open access 03 November 2022 IMPROVING ACCURACY AND REPRODUCIBILITY OF MASS SPECTROMETRY CHARACTERIZATION OF PROTEIN CORONAS ON

NANOPARTICLES Article 11 June 2025 MAPPING AND IDENTIFICATION OF SOFT CORONA PROTEINS AT NANOPARTICLES AND THEIR IMPACT ON CELLULAR ASSOCIATION Article Open access 10 September 2020

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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). AUTHOR INFORMATION Author notes *

Dominic Docter and Ute Distler: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * Molecular and Cellular Oncology, ENT, University Medical Center of Mainz, Mainz,

Germany Dominic Docter, Desirée Wünsch, Angelina Hahlbrock & Roland H Stauber * Institute for Immunology, University Medical Center of Mainz, Mainz, Germany Ute Distler, Wiebke Storck, 

Jörg Kuharev & Stefan Tenzer * Institute for Molecular Biology, University Duisburg-Essen, Essen, Germany Shirley K Knauer Authors * Dominic Docter View author publications You can also

search for this author inPubMed Google Scholar * Ute Distler View author publications You can also search for this author inPubMed Google Scholar * Wiebke Storck View author publications You

can also search for this author inPubMed Google Scholar * Jörg Kuharev View author publications You can also search for this author inPubMed Google Scholar * Desirée Wünsch View author

publications You can also search for this author inPubMed Google Scholar * Angelina Hahlbrock View author publications You can also search for this author inPubMed Google Scholar * Shirley K

Knauer View author publications You can also search for this author inPubMed Google Scholar * Stefan Tenzer View author publications You can also search for this author inPubMed Google

Scholar * Roland H Stauber View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS 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) RIGHTS AND PERMISSIONS Reprints and

permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Published: 31 July 2014 * Issue Date: September 2014 * DOI: https://doi.org/10.1038/nprot.2014.139 SHARE THIS

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