*2.1.5. Label-free MS/MS quantification*

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 .

Isothermal titration calorimetry is a method that allows the determination of thermodynamic parameters in a solution (binding affinity, binding stoichiometry, and binding enthalpy change [33]), in particular the ones coming from interactions between biological macromolecules [42]. This process is based on the changes in heat caused by the protein adsorption to the nanoparticle [43]. The main advantage of this method relies on the fact that it allows the characterization while still in the incubation medium [43], which consequently allows a

UV-visible spectrometry is a process based on the ratio between the passed light measured and the incident light in the UV-visible wavelength [33]. The presence of the protein corona induces changes on the absorption spectrum [17], which makes it an easy, fast, and applicable approach, as it requires no other chemicals or resources other than the protein corona itself [33]. However, it is an unreliable method, as the radiation energy reaching the sample is low [44]; it is very influenced by parameters such as size, temperature, pH [33], and equipment errors, which have a much bigger impact, as there is no other chemical or technique applied

SILAC, an acronym to stable isotope labeling by amino acids in cell culture, is a procedure where an essential a.a. has been replaced by its stable isotope counterpart in the cells' growth medium, making this "heavy" amino acid incorporated into all expressed proteins [45]. This causes the growth of two populations of cells: the ones growing in "light" medium containing the natural isotope in the amino acids and the ones growing in "heavy" medium containing stable isotope-labeled amino acids [33, 46]. After complete labeling, equal amounts of labeled and unlabeled cells or protein extracts are mixed in the cell population. The samples are then digested into peptides and then analyzed with mass spectrometry. The quantification of SILAC is thus based on the ratio of introduced isotope-labeled peptides to unlabeled peptides [46]. The many advantages of SILAC are its easy implementation, reasonable quantitative

*2.1.1. Isothermal titration calorimetry (ITC)*

greater optimization of the nanoparticle.

to lower the risk of incorrect results [44].

accuracy, and high reproducibility [46].

*2.1.3. Stable isotope labeling by amino acids in cell culture (SILAC)*

*2.1.2. UV-visible spectrometry*

32 Protein-Protein Interaction Assays

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 information, with great reproducibility [49].
