*2.2.8. X-ray crystallography*

get sorted by molecular weight [53], and staining is needed with an appropriate pigment, for instance, Coomassie blue [54]. The molecular weight is then compared with the one shown by the markers, and a densitometry analysis is performed [33]. This procedure is suitable to characterize the proteins that form the corona, comparing the proteins obtained from the plasma with the ones that are found in the corona. It is thus possible to verify exactly which ones get adsorbed to its surface and, therefore, the ones that have greater affinity to the nanoparticle [43].

Fluorescence spectroscopy is a method that allows the measurement of the fluorescence of a compound, when excited at a given wavelength [33]. The fluorescence may come from the protein (intrinsic probes), the nanoparticle, or even a fluorophore added to the complex (extrinsic probes) [33, 55], which will be picked up by the amino groups and then detected in

Shotgun proteomics is a widely used technique in proteomics for identifying proteins [57], with great sensitivity, making it a great influence in the discovery of clinically actionable biomarkers [58]. To perform it, a complex mixture of proteins is separated by sequence-specific proteolysis, forming peptides that will then be separated in smaller fragments to be later analyzed by mass spectrometry [57]. Each peptide will have a mass associated to it, but since peptides may have the same a.a., but in a different arrangement, it is important to assess the sequence, which can be done by the ratios of its mass spectrum, that is, by MS/MS [57].

This method is often disregarded by researchers, due to its early problems in reproducibility and fallible nature [58, 59]. However, the development of bioinformatic tools has allowed a

One of the ways frequently used in replacement of shotgun MS/MS is selected reaction monitoring (SRM) [60], also known as multiple reaction monitoring (MRM). This assay makes use of a triple-quadrupole mass spectrometer [60], where peptides from a previously digested protein go through. In the first analyzer, molecular ions with similar mass to the peptide are selected, followed by fragmentation of the peptide bonds in the second analyzer. Lastly, in the third and final analyzer, the fragmented ions from the peptide pieces are measured, originating a transition signal [61]. This method does not record any full-mass spectrum, increasing its sensitivity and allowing the detection of scarce proteins in complex

FTIR is a procedure that gives information about the surface properties of the nanoparticle and the protein corona, as it allows the detection of its attachment [33]. Standard Raman

decrease in these problems, making it a viable option for proteomic research [57–59].

*2.2.5. Selected reaction monitoring (SRM)/multiple reaction monitoring (MRM)*

*2.2.6. Fourier transform infrared and Raman spectroscopy*

*2.2.3. Fluorescence spectroscopy*

34 Protein-Protein Interaction Assays

the fluorescence spectrometer [56].

*2.2.4. Shotgun MS/MS*

mixtures [60].

Considered one of the primary sources of structural information about the protein-ligand complex [67], and it is based on the positions and intensities of the reflections as measured in the diffraction pattern of the crystal [67]. This method has big challenges associated to it, as the quantity of radiation needed may be excessive and cause damages to the proteins before a signal is obtained [68], and there are many uncertainties when applying it, such as identity or location of the proteins to be evaluated [67].
