Effective Lock-in Strategy For Proteomic Analysis Of Corona Complexes Bound To Amino-free Ligands Of Gold Nanoparticles

BackgroundAs an important class of materials,gold nanoparticles?GNPs?have the advantages of a non-cytotoxic nature,chemical stability,easy synthesis,and functionalization.GNPs have recently shown great promise in nanomedicine for both diagnosis and therapy.For a specific application,gold nanoparticles?GNPs?are commonly functionalized with various biological ligands,including amino-free ligands such as amino acids,peptides,proteins,and nucleic acids.Upon entering a biological fluid,the protein corona formed around GNPs can conceal the targeting ligands and sterically hinder the functional properties.Previously,the protein corona was prepared routinely by standard centrifugation or sucrose cushion centrifugation.However,such methodologies are not applicable to the exclusive analysis of a ligand-binding protein corona.Therefore,we urgently need to propose a new method to enable more stable binding of GNPs to amino acid-free ligands and to analyze the resulting protein corona complexes in detail.Methods1.Cysteine-modified GNP synthesis and characterization:The chloroauric acid?AuCl₄·4H₂O?was solubilized in ultrapure H₂O.Trisodium citrate dehydrate and L-cysteine were added sequentially and under certain conditions,GNPs in water were obtained via filtration,and then characterized using transmission electron microscopy?TEM?.2.Collection of Human Blood Plasma Samples:Blood samples were harvested in collection tubes with ethylenediaminetetraacetic acid by venipuncture and immediately centrifugation.3.Formation and Separation of the Protein Corona of GNPs:The appropriate volumes of GNPs and plasma were performed at 37°C with shaking to allow for protein association.There methods were applied to separate the protein corona during this experiment,Method I was the standard centrifugation method and Method II was the sucrose cushion centrifugation method.In our new method?Method III?,after incubation of GNPs with plasma,quick fixation was initially performed using formaldehyde and explored formaldehyde concentration and reaction time.At the most condition of the formaldehyde concentration and reaction time,we got the protein corona after the cysteine-binding protein corona was effectively separated from unbound proteins by stringent washing.4.Qualitative and quantitative analysis:The protein solution obtained by the three methods was boiled,in which the cross-linked formaldehyde sample was boiled for 20 minutes to destroy the chemical cross-links between proteins,and the protein sample was concentrated in a short gel using SDS-PAGE.After the enzymatic hydrolysis in the gel,the liquid mass was used.Mass spectrometry?LC-MS/MS?was used to detect the peptides of the enzymolyzed peptides.The bioinformatics analysis and statistical analysis were performed after protein searches.The date of the three methods were compared by non-standard quantitative methods.5.The formaldehyde-linked site analyses of protein corona:Cross-linked samples of unboiling formaldehyde were separated by SDS-PAGE,slices were excised and subjected to in-gel digestion,followed by mass spectrometry detection.Variable modifications were set up in the Mascot search where cysteine markers were used as variable modifications for site resolution.Results1.The results of Transmission electron microscopy?TEM?show that the size of cysteine-attached gold nanoparticles were 17 nm.2.When explored formaldehyde concentration and reaction time after the protein corona complexes of the GNPs with amino-free ligands obtained,We found that 5-s crosslinking with 7.5%formaldehyde was achieved for rapid fixation of the cysteine-binding protein corona.3.295±12,298±20,and 217±5 proteins were identified by MS from the coronas obtained using Methods I,II,and III,respectively.And all the proteins from the three methods displayed similar MW,pI,and GRAVY distribution profiles.The cysteine-binding corona proteins from Method III had distinct physicochemical profiles and physiological molecular functions relative to the whole corona from Methods I and II.These results indicated that the Method III-prepared corona could serve as a specific cysteine-binding protein corona4.A total of 456 proteins detected in at least two of the three preparations were identified from the protein corona complexes.There had302 nodes and 8572 interactions were exported into the database Search Tool for the Retrieval of Interacting Genes/Proteins?STRING?.The results showed that the protein corona complexes were a huge network of protein interaction.We also found that 11 proteins were detected to be directly interacted with cysteine and it provided the direct evidence for covering up the intended functionalization of the GNP ligand.ConclusionIn this study,the presented methodology catches the corona proteins interacted with amino-free ligands of GNPs in time and provides a generic way to analyze a nanoparticle corona bound to amino-free ligands.It also provides a methodological tool and the direct evidence for covering up the intended functionalization of the GNP ligand.