**2. Characterization of protein corona by proteomic strategies**

Since the formation of the protein corona has a great impact on the nanoparticle's performance when applied to a biological system, it is important to assess its structure and composition, in order to minimize the adverse effects it may have on the nanoparticle's use. Any alteration in shape, size, electron transfer, or others may come from the binding of the protein corona to the nanoparticle and may be used as parameters of comparison to be tested between nanoparticles, before and after administration to a biological fluid [29]. However, it is necessary to separate the nanoparticle-protein complex from the excess of plasma proteins [30] before assessing the composition of the protein corona. This is frequently made by centrifugation [30, 31], but it can have many adverse effects in the corona, due to the alterations caused by washing steps as well as gradient and volume variations [30, 32, 33]. In order to avoid loss of proteins from the corona, or even tainting the protein corona sample with the proteins in excess from the plasma, centrifugation can be accompanied by other procedures, such as size exclusion chromatography [32] or microfiltration [33, 34]. In the case of magnetic nanoparticles, a one-step centrifugation does not work, as it agglomerates the nanoparticles, making it necessary to perform a magnetic separation [35, 36]. As said by Megido et al. [33], the main methods of evaluation can be held as qualitative or quantitative, being summarized in **Figure 1**.
