Capillaire elektroforese massaspectrometrie benaderingen voor karakterisering van het eiwit en metaboliet corona verworven door nanomaterialen

This protocol is the first to implement CESI-MS for the characterization of not only the protein corona, but also the metabolite corona of nanomaterials. CESI offers a orthogonal separation to traditional LCMS methods, which is both highly reproducible and sensitive while using just a few nanoliters of sample. To begin, split two milliliters of the human plasma into one milliliter aliquots, one for the metabolite and protein corona, and one for the plasma metabolome characterization.

Incubate one milligram per milliliter of nanomaterials in the human plasma for one hour at 37 degrees Celsius while mixing gently at 500 rotations per minute in a thermo mixer. Then, pellet the nanomaterials by centrifuging at 4, 000 times G for 15 minutes at four degrees Celsius. Collect the plasma supernatant for metabolite corona analysis and retain the nanomaterial protein corona complex for protein corona characterization.

Resuspend the pellet in one milliliter of 10X PBS buffer, and vortex vigorously for two minutes to remove unbound proteins. Centrifuge the solution at 4, 000 times G for 15 minutes at four degrees Celsius to pellet the nanomaterials, then remove the supernatant carefully without disturbing the pellet. Resuspend the pellet in one milliliter of ABC buffer, and vortex vigorously for two minutes to remove unbound proteins and unbound salts.

Repeat the centrifugation step and discard the supernatant. Dissolve the pellet in 20 microliters of ABC buffer containing 10 millimolar dithiothreitol to reduce protein disulfide bonds. Incubate the solution for 30 minutes at 56 degrees Celsius.

Add two micrograms of sequencing grade trypsin to the 20 microliters of ABC buffer containing 0.1%surfactant, and incubate the digest solution at 37 degrees Celsius for 16 hours. Alkylate the sample by adding 20 microliters of 55 millimolar iodoacetamide in 100 millimolar ABC and incubate at room temperature for 20 minutes. Add 20 microliters of 0.1 molar hydrochloric acid to cleave the surfactant and leave the sample for 10 minutes at room temperature.

Enrich and de-salt the peptides using a C18 packed desalting pipette tip as described in the text manuscript. Then lyophilize the sample and store it at negative 20 degrees Celsius until analysis. Take the control plasma and the supernatant from the nanomaterial plasma incubation and put them on ice.

Aliquot 50 microliters from each sample into separate vials, and dilute tenfold with distilled water. After vortexing, move 50 microliters of this diluted sample to new vials. Add 200 microliters of chloroform, 250 microliters of methanol, and 350 microliters of distilled water and vortex vigorously for two minutes.

Centrifuge at 20, 800 times G at four degrees Celsius for 10 minutes. Take 500 microliters of the supernatant and filter it through a three kilodalton centrifugal filter for two hours at 10, 000 times G at four degrees Celsius. Dry 430 microliters of the ultra filtrate in a speed vac and freeze the sample.

Prior to installation of the capillaries into the CESI instrument, check that the inlet and outlet ends of the capillaries are intact and that there are no visible breakages in the capillary. Then place a new neutral coded capillary into the instrument, following the manufacturer guidelines. Flush the separation capillary in the forward direction with 100 millimolar hydrochloric acid, then with BGE, and finally with distilled water, using the conditions described in the text manuscript.

Then flush both the separation and conductive capillary with BGE followed with distilled water, 100 millimolar hydrochloric acid, distilled water again, and finally with BGE. Couple CESI to the MS using the nano spray source and adapter. For protein corona analysis, resuspend the lyophilized samples in 20 microliters of 50 millimolar ammonium acetate at pH four.

Perform CESI-MS analysis for the samples as described in the text manuscript. Collect mass spectra data between 250 and 2, 000 m/z, with top 10 data dependent fragmentation acquisition, and rinse the capillary between samples. Place a new bare fused silica capillary into the CESI instrument.

Flush the separation capillary in the forward direction using 100%methanol, then flush both the separation and conductive capillary with BGE to ensure droplet formation at the outlet ends. Then flush the capillary with distilled water, followed by 0.1 molar sodium hydroxide, distilled water again, and finally with BGE. Couple CESI to the SEMS using the nano spray source and adapter.

To perform metabolite corona analysis, resuspend the nanomaterial exposed and unexposed plasma samples in 430 microliters of distilled water and vigorously vortex for two minutes. Filter the samples through a 0.1 micrometer membrane filter. Add five microliters of the internal standards, as mentioned in the text manuscript, to 95 microliters of the filtrate and vortex vigorously.

Centrifuge at 16, 100 times G and four degrees Celsius. Perform CESI-MS analysis for the samples as described in the text manuscript. The use of CESI-MS for proteomic and metabolomic separation and detection demonstrate good separation windows on both capillaries for each approach, enabling a comprehensive characterization of the biomolecular corona.

The proteomic CESI-MS method is capable of distinguishing between an array of proteins and protein concentrations in the corona across a wide range of nanomaterial compositions, and can distinguish a unique protein corona for each nanomaterial. This CESI-MS metabolomics approach enables a quantitative analysis of the metabolite corona, and can be used to uncover its unique fingerprints. Although this approach passively characterizes the nanomaterial metabolite corona, it is still capable of uncovering interesting insights into the role of metabolites in the biomolecular corona, such as the differential absorption of isomers.

This technique makes it possible to characterize both the protein and the metabolite corona, allowing for improved understanding of how the complete biomolecular corona affects nanomaterial uptake by cells.