Mass spectrometric analysis of the protein corona composition

In order to have a more comprehensive understanding of the processing of nanoparticles in the intracellular environment, the composition of the protein corona must be analyzed.

For this purpose, mass spectrometric approaches can be used.

Mass spectrometry is based on the analysis of ionized species. The ion sources are adapted to the analytical approach: in elemental analysis, hard ionization, e.g., an inductively coupled plasma ion source is used. When the composition of larger molecules is in the focus of the study, soft ionization is applied, e.g., by an electrospray ion source (ESI), which is introduced in more detail below. The ionized species are separated in a mass analyzer based on their mass-to-charge ratio. There is a variety of mass analyzers that apply either an electric field, a magnetic field, or their combination in a static or dynamic manner. Based on the dynamics of charged particles in an electromagnetic field [159, 160], the mass-to-charge ratio (m/Q) determines the motion of the ion, and the ion can be identified once its initial conditions are known. In most cases, instead of the m/Q ratio, the dimensionless m/z ratio is used to characterize an ion, where z is the elementary charge number of the ion proportional to the charge of a proton [159, 160].

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For proteins and peptides, soft ionization techniques are used, e.g., matrix-assisted laser ionization/desorption (MALDI) or ESI. These ionization techniques prevent the sensitive biomolecule chains from fragmentation. In this thesis, ESI was used to convert the peptides separated by liquid chromatography from liquid phase into charged species in the gas phase. In ESI, ionization occurs when a solution passes through a needle with high voltage (4-5 kV) compared to a counter electrode [160]. If the solution is an electrolyte, it receives a positive or negative net charge and transforms into a fine charged mist at the end of the needle. A flow of dry gas passing in the opposite direction allows for the evaporation of the solvent in the droplets, and as a result of the identically charged ions getting close to each other, the arising Coulombic repulsive forces aid the formation of much smaller droplets. This process repeats itself until the charged ions become completely solvent-free, which eventually flow into the mass analyzer.

The instrument used in this thesis work was an impact II UHR-Qq-TOF-MS (ultrahigh-resolution double-quadrupole time-of-flight mass spectrometer). In this tandem-MS instrument, there are two quadrupoles (Qq) and a time-of-flight mass analyzer (TOF).

Quadrupoles consist of four parallel metal rods positioned symmetrically, and each pair of opposing rods is electrically connected. In the first quadrupole, a radio-frequency voltage with a DC offset is applied to the pairs of rods, resulting in an electric field that destabilizes the trajectories of most ions and only allows a specific m/z range to pass through the analyzer [160]. The second quadrupole acts as a collision cell, where only radio-frequency voltage is applied with no m/z filtering, and the ions colliding with the applied inert gas flow (e.g., N2) undergo fragmentation [160]. The fragments are then analyzed by the TOF mass analyzer, where a known electric field is applied to accelerate ions, resulting in the same kinetic energy for ions with identical charge [160]. Therefore, the velocity of identically charged ions, and their time-of-flight, solely depends on their mass, heavier ions reaching the detectors later than the lighter ions.

A tandem-MS experiment of a mixture of peptides can yield large datasets, which are difficult to evaluate manually. Nowadays, tandem-MS data can be analyzed by readily available commercial softwares specialized to the analytes of interest. In this thesis, commercially available softwares and open databases were used to identify peptides and determine the corresponding protein species, as described in Chapters 7 and 9.

21 Most of the studies of the protein corona found in the literature begin with isolating the nanoparticle-protein bioconjugates, which is generally followed by the electrophoretic analysis of the corona constituents. The purification of the bioconjugates is often based on centrifugation, including differential centrifugation [36] and the use of a sucrose cushion [35], while the proteins are generally identified eventually with the help of mass spectrometry [32, 35, 36, 161, 162].

The formation of the protein corona is extensively studied in in vitro solutions. Tenzer and colleagues demonstrated that the size of the nanoparticles was the most deterministic trait of nanoparticles in the formation of the protein corona [163]. The group further discussed that the protein corona forms rapidly once the nanoparticle is introduced into the biomolecular system, and the building proteins do not change over time, only their relative concentration to each other [32]. The quantitative analysis of the protein corona is difficult due to the extensive purification of the nanoparticle-protein bioconjugates, but attempts have been made for the protein enumeration, for example, based on the average number of sulfur atoms in proteins with ICP-MS [35].

The proteomic analysis of the adsorbed proteins on nanoparticles in cell lysates can help approximate the expected interactions in live cells. It was found that in such an in vitro environment, different proteins interacted with the nanoparticles from those expected based on the surface functionalization of nanoparticles, and the adsorption of several immunoproteins can reflect on the biocompatibility of the used nanoparticles [37]. These results support that the engineered surface properties only influence the protein adsorption up to a certain extent, as discussed earlier. The possibilities of the therapeutic application of gold nanoparticles were demonstrated in experiments where cancer-specific proteins were found enriched in the protein corona even without the active processing of cells [34, 38].

Regardless of the extensive studies of the protein corona composition in different protein solutions, the intracellular formation of the protein corona is still poorly understood.

Interestingly, the proteome of the cellular compartments in which internalized particles reside has been investigated for decades now [82, 83], but the protein corona formed inside the cells has still not been directly analyzed. The exchange of fluorescently labeled proteins was observed on the surface of intracellular nanoparticles, and the total intensity

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of fluorescence informed about both the residence time of the nanoparticles in the cells and the cleavage of the serum proteins [69]. The intracellular protein corona composition was evaluated in rainbow trout gill cells, and with differential centrifugation, the protein corona complex was recovered, containing both hard and soft corona proteins [36].

Despite the significant advances in understanding the intracellular protein corona formation, these results yielded a list of proteins that may or may not interact directly with the nanoparticles’ surface as a cellular response to their presence, and therefore, further studies are required for the analysis of the hard protein corona formed inside cells.

2.7. Cryo soft X-ray nanotomography in the studies of the cellular ultrastructure