**2.1. Quantitative proteomics**

of characteristics in order to be considered as good theranostic agents, such as suitable size [6] and shape [7] for cell penetration, biocompatibility, surface charge, efficient targeting [8], and fluorescence, among others [9]. Despite of these advantages and promising applications, there are still many problems associated to the entrance of the nanoparticle in a physiological environment, which may be justified with different intrinsic characteristics of the nanoparticles [10]. In general, there are two different nanoparticle identities, such as "synthetic identity," which refers to their intentional physicochemical properties [11], and a "biological identity," which is related to the physicochemical properties shown by the nanoparticle after its application in a physiological environment and interaction with the presented biomolecules [10]. This "biological identity" is profoundly related to the formation of the protein corona, as it significantly alters the size, shape, and surface charge of the nanoparticle [12, 13]. The protein corona is formed after the entrance of the nanoparticle in a physiological environment, such as the bloodstream and/or peripheral blood, where the presence of thousands of proteins [14] (among other biomolecules) causes their adsorption onto the nanoparticle surface [15], in a corona shape [16]. The formation of this corona is energetically favorable, with a decrease of enthalpy and increase of entropy [15]. It can be divided into two categories: a "hard" corona, and a "soft" corona. The "soft" corona is based on abundant proteins that firstly bind to the nanoparticle through low-affinity bonds, and the "hard" corona is more dense [17], based on sparse proteins that replace the "soft" proteins over time, due to their higher affinity bonds [11], which is known as the Vroman effect [18]. The composition of the corona is directly dependent on the biomolecular composition of the physiological environment that surrounds it [19, 20], the time of exposure [11, 17, 21], and also incubation conditions (such as temperature or mild stirring), among others. Moreover, it is clear that the protein corona is not static and varies in the course of time; in other words, it's dynamic [17, 21]. Eventually, it will reach a state of equilibrium, steady stochastic state, where the association and dissociation rates for each protein occur equally [21], unless it is further incubated in a different biological media or proximal biological fluid, with a formation of a new corona [22]. The great majority of the proteins that form the corona are independent of the size and surface charge of the nanoparticle but are very influenced by the chemical properties of the material that constitutes the nanoparticle [23]. However, there are still sensitive proteins to size and surface charge, whose nature can change and consequently alter the interactions between nanoparticles and cells with the consequent alteration of the biological outcome and biological impacts [23]. For instance, if opsonins bind to the nanoparticle in the protein corona, they will be recognized as a "threat," and consequently are phagocytosed by macrophages [24]. It is crucial to prevent opsonization, "camouflage" the nanoparticle to avoid the phagocytosis, and keep the nanoparticles in circulation, which can be achieved by the application of a polymer coating, such as poly(ethylene glycol) (PEG) [25]. This polymer coating also prevents the formation of the protein corona, which can later compromise the nanoparticle internalization by the cells [25]. It is thus important to study the influence of the protein corona in the internalization of the nanoparticle, as the interactions of the nanoparticles with cells in in vivo studies are much different from the in vitro ones [26–28], which can prove to be an obstacle in the generalized application of this ther-

30 Protein-Protein Interaction Assays

anostic approach based on nanomaterials.

Quantitative proteomics is the collection of techniques that allow the determination of the number of proteins in a sample, which may be its absolute amount or just the relative change

in amounts between two states [37]. There are many problems associated to the methods used for these assays, such as difficulties in reproducing the results and lack of precision in the measurements [38, 39], but recent technologies have allowed to minimize such issues [40], increasing the depth and coverage [38], which can also be done by using several techniques simultaneously and by defining standards for reproducibility [39]. The most commonly used assays are isothermal titration calorimetry (ITC), UV-visible spectrometry, stable isotope labeling by amino acids in cell culture (SILAC), isobaric tag for relative and absolute quantitation (iTRAQ), and label-free MS/MS quantification. There are also quantitative approaches that make use of two-dimensional electrophoresis (2-DE) [41], but it's mostly used for qualitative proteomics [39], due to the current limitations in performing quantitative assays .

*2.1.4. Isobaric tag for relative and absolute quantitation (iTRAQ)*

*2.1.5. Label-free MS/MS quantification*

tion, with great reproducibility [49].

**2.2. Qualitative proteomics**

*2.2.1. Circular dichroism*

mation [51].

*2.2.2. SDS-PAGE*

Isobaric tag for relative and absolute quantitation, also known as iTRAQ, is a widely used method in proteomics for quantification. It is based on mass spectrometry (MS) [47] and is useful in situations where the proteins come from different sources in the same sample [33]. This technique makes use of amine-reactive reagents with different isotopic masses between them [47], labeling the peptides differently and allowing for a clear distinction when analyzing MS scans, as various peptides appear each in a single peak [33]. However, this method has a great disadvantage concerning its cost [48], which makes it impracticable when compared to cheaper alternatives.

Protein Interactions and Nanomaterials: A Key Role of the Protein Corona in Nanobiocompatibility

http://dx.doi.org/10.5772/intechopen.75501

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Label-free quantification methods make no use of labeling on the proteins, relying only on the measurement of ion intensity changes in chromatography or on spectrum counting of fragments of peptides in a given protein [46]. This procedure is especially suited for biomarker discovery in large sample sets, as it is not needed labeling in any protein [49]. Labeling also limits the dynamic range, resulting in loss of signal and possible omission of proteins [50]. Therefore, using a label-free quantification approach allows the gathering of reliable informa-

Qualitative proteomics refers to the assays designed for identification of proteins in a sample and are often performed not only for identification but also for quantitative purposes, such as the abovementioned 2-DE electrophoresis [39]. Other assessments that allow identification of proteins are circular dichroism (CD), SDS-PAGE, fluorescence spectroscopy, shotgun MS/MS, selected reaction monitoring (SRM)/multiple reaction monitoring (MRM), Fourier transform infrared and Raman spectroscopy, nuclear magnetic resonance (NMR), and X-ray [33].

Circular dichroism is an assay based on the determination of the secondary structure, folding and binding properties of proteins [51], using the difference between the absorption of left and right circularly polarized light [52]. This method is based on the optic properties shown by the conformation of the protein, which can be altered when interacting with a nanoparticle. The nanoparticle itself shows no influence in the light, as it is not a chiral compound [33], and it can also be used with small amounts of proteins (20 μg) in a short amount of time [51], making it a viable way of assessment. However, it has some limitations, such as unfeasibility with complex mixtures of proteins [33] and impossibility in obtaining residue-specific infor-

One of the most used methods in proteomics, electrophoresis, is a procedure that separates proteins in a sample according to their charge. Using a gel of polyacrylamide, a protein solution is applied, and in relation to their charge, proteins will migrate across the gel [53]. The proteins
