**1. Introduction**

326 Gel Electrophoresis – Advanced Techniques

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#### **1.1 Nanoparticles**

Adverse health effects have been associated with the exposure to particulate matter (PM) ever since the London smog in the winter of 1952. Recent estimates attribute about 12,000 excess deaths to have occurred because of acute and persisting effects of the London smog (Bell & Davis, 2001). Over the years a number of epidemiological studies have shown that PM from combustion sources such as motor vehicles contributes to respiratory and cardiovascular morbidity and mortality (Kreyling et al., 2002, 2006, Wick et al., 2010). Especially so do the ultra-fine particles (UFPs) with a diameter less than 0.1 micrometer. UFPs from combustion engines are capable to translocate over the alveolar–capillary barrier (Rothen-Rutishauser et al., 2007). When nano-sized PM (nanoparticles, NP), which are small enough to enter the blood stream, do so they are likely to interact with plasma proteins and this protein-NP interaction will probably affect the fate of and the effects caused by the NPs in the human body. Herein we present results showing that several proteins indeed are associated to NPs that have *in vitro* been introduced to human blood plasma.

NPs are atoms and molecules defined as particles less than 100 nanometers in at least one dimension (Elsaesser & Howard, 2011). Due to the plethora of NPs being produced in various forms (e.g. spherical, fibers, rods, clusters) or by different processes (e.g. flamespray synthesis, chemical vapor deposition), defining the characteristics of a NP is not an easy task even when it comes to manufactured NPs and when considering those formed unintentionally during processes such as combustion in motor vehicles it becomes an even harder task. This variation in properties according to the respective composition of NPs is also the basis of the wide range of potential applications, from medicine to consumer products. Due to the unique physicochemical properties of nanomaterials, there are plenty of possibilities for NPs to enter the human body, either deliberately as medicines or unintentionally as environmental contaminants and thus potentially cause adverse human health effects (Elsaesser & Howard, 2011; Stern & McNeil, 2008). Although many characteristics have been highlighted as driving the potential adverse health effects associated with NP exposure, it has been specifically the size and increased surface area of NPs that has been concluded as elucidating any such adverse effects observed (Elsaesser & Howard, 2011; Stern & McNeil, 2008).

Two-Dimensional Gel Electrophoresis and Mass

**1.2 General introduction to the 2-DE technique** 

improve the approach.

toxicology.

Spectrometry in Studies of Nanoparticle-Protein Interactions 329

generalizable conclusions and should therefore be given ample attention. Furthermore, most of these toxicological parameters involve proteins and/or actions carried out by proteins. Thus it is pivotal to the understanding of NP toxicology, and thereby the possibility to predict health effects caused by NPs, to understand NPs interactions with proteins. One of the best, if not the best, technique to separate proteins is two-dimensional gel electrophoresis (2-DE). Preferably, this is combined with peptide mass fingerprinting and MALDI-TOF MS analyses for fast identification of the separated proteins, which then may be followed by tandem MS analyses for sequence information. Here we present our results regarding serum protein interactions with metal-nanoparticles (Al2O3, ZnO/Al6%, SiO2) and Carbon Nanotubes, obtained using 2-DE and MS. Furthermore, we elaborate on and exemplify different aspects of the 2-DE/peptide mass fingerprinting-technique to further

Two-dimensional gel electrophoresis (2-DE) is an excellent method for separation of proteins from most kinds of tissues and complex mixtures of proteins (O`Farrel-1975). Both qualitative characterization of the protein expression, including post-translational modifications and quantitative characterization comparing the protein expression in different individuals or groups, are possible by this technique. Two steps are included, the isoelectric focusing (IEF) step, where the proteins are separated according to their isoelectric point (pI) in a pH-gradient, and the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) step, where the proteins are separated according to their molecular weight. Since it is less common that two proteins have the same isoelectric point and molecular weight, this will result in each protein migrating to its own unique position. The 2-DE technique allows, depending on the nature of the sample, the separation of 500- 3000 protein spots and the resolution can be improved, e.g. by removal of abundant proteins or by composite gels from overlapping pH-gradients. Proteins separated by gel electrophoresis can be visualized by a number of methods using different types of stains. Various stains interact differently with the proteins and some of the stains used are not even specific for proteins. The degree of sensitivity is also different. Processing data from stained protein gels by computers includes the gel images being digitized by an imaging system and then analyzed using computer software allowing a number of different measurements such as number, size, and intensity of the stained protein spots. Separated proteins are then identified by mass spectrometry (MS). The proteins are in-gel digested and extracted peptides analyzed by peptide mass fingerprinting or peptide sequencing. Two widely used MS instruments used for these respective analyses are matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) where peptides are transferred from solid phase to gas phase, and electrospray ionization tandem mass spectrometry (ESI MS/MS) where peptides are transferred from liquid phase to gas phase. The key advantage with 2-DE is the ability to separate protein isoforms. On the other hand, very large and hydrophobic proteins are underrepresented in 2-DE and the need of peptide extraction from the in-gel digests may influence to amounts of analytes available for MS protein identification. Nevertheless, combining the separation and analytical ability of the 2- DE technique with the identification power of MS provides a powerful tool in human

UFPs from combustion sources, such as motor vehicles, are capable to promote atherosclerosis, thrombogenesis and other cardiovascular events mainly via the ability to induce inflammatory and protrombotic responses (McAuliffe & Perry 2007). Thus, NP induced effects within the lung has been studied over the past twenty years (Mühlfeld et al., 2008; Rothen-Rutishauser et al., 2007). Indeed, exposure to most forms of NPs will initially be via inhalation, especially when considering occupational exposure, and thus will affect the respiratory system. The respiratory system is by far the main port of entry even though the gut and skin are also possible ways of entry. However, NP localization and fate is not only restricted to their portal of entry. NPs can be distributed to organs distal to their site of exposure, so that potential NP toxicity can occur in any secondary site. Research into the possible secondary toxicity of NPs is quite limited. Even so studies investigating the effects of NP translocation to secondary organs have shown that NPs can elicit negative effects to the liver, brain, GI tract (following inhalation), spleen, reproductive systems and the placenta (Kreyling et al., 2002, 2006; McAuliffe & Perry 2007; Wick et al., 2010). NPs toxic mechanisms at the cellular level includes protein misfolding and protein fibrillation causing major problems in the brain and chronic inflammation as a result of nanoparticle exposure, for example in the lung and other organs, via frustrated phagocytosis or production of reactive oxygen species. A vulnerable target for possible toxicological effects of nanoparticles is the fetus. Gold nanoparticles have been shown to cross the maternal-fetal barrier and fullerenes were found to have a fatal effect on mouse embryos (Elsaesser & Howard, 2011; Stern & McNeil, 2008).

When nanoparticles enter the blood vessels, or any biological fluid, e.g. saliva or mucus they are most likely surrounded by a layer of proteins. This dynamic protein "corona" depends on the concentration in the biological fluid and hence the composition of the layer varies in different parts of the body (Cedervall et al., 2007; Lundqvist et al., 2008; Lynch et al., 2007; Walczyk et al., 2010,). Thus, the reactions in the body to such a NP-protein complex is most likely different from that induced by the bare NP and possibly affecting biodistribution and thereby causing unwanted side effects (Adiseshaiah et al., 2010; Leszczynski 2010,). The function of protein coating is not fully known but since most nanoparticles show strong affinity for proteins, it is of importance to investigate this interaction in different fluids. In blood, plasma proteins constituting the NP corona is possibly affecting a wide range of effects such as phagocytosis via immunoglobulins and complement (Dobrovolskaia & McNeil 2007), coagulation via prothrombin (Dobrovolskaia 2009) and the distribution of lipoproteins (Benderly et al., 2009; Hellstrand et al., 2009; Zensi et al., 2010).

The availability and toxicity of any substance to a biological organism is determined by both the concentration/dose that the organism is exposed to, as well as the "toxicokinetics" of the substance. These include the uptake, transport, metabolism and sequestration to different compartments by the organism, as well as the elimination of the substance from the biological organism. These parameters are essential since the potential toxicity of substances is dependent upon the specific organs or cell types exposed, which form the substance is in (e.g. bound to serum protein, aggregated, dissolved, oxidized), as well as the period of time the substance interacts/remains at the site of primary and secondary exposure. These parameters are influenced by the physical–chemical characteristics of the substance, therefore a detailed characterization of the substance is pivotal in order to allow

UFPs from combustion sources, such as motor vehicles, are capable to promote atherosclerosis, thrombogenesis and other cardiovascular events mainly via the ability to induce inflammatory and protrombotic responses (McAuliffe & Perry 2007). Thus, NP induced effects within the lung has been studied over the past twenty years (Mühlfeld et al., 2008; Rothen-Rutishauser et al., 2007). Indeed, exposure to most forms of NPs will initially be via inhalation, especially when considering occupational exposure, and thus will affect the respiratory system. The respiratory system is by far the main port of entry even though the gut and skin are also possible ways of entry. However, NP localization and fate is not only restricted to their portal of entry. NPs can be distributed to organs distal to their site of exposure, so that potential NP toxicity can occur in any secondary site. Research into the possible secondary toxicity of NPs is quite limited. Even so studies investigating the effects of NP translocation to secondary organs have shown that NPs can elicit negative effects to the liver, brain, GI tract (following inhalation), spleen, reproductive systems and the placenta (Kreyling et al., 2002, 2006; McAuliffe & Perry 2007; Wick et al., 2010). NPs toxic mechanisms at the cellular level includes protein misfolding and protein fibrillation causing major problems in the brain and chronic inflammation as a result of nanoparticle exposure, for example in the lung and other organs, via frustrated phagocytosis or production of reactive oxygen species. A vulnerable target for possible toxicological effects of nanoparticles is the fetus. Gold nanoparticles have been shown to cross the maternal-fetal barrier and fullerenes were found to have a fatal effect on mouse embryos (Elsaesser & Howard, 2011; Stern &

When nanoparticles enter the blood vessels, or any biological fluid, e.g. saliva or mucus they are most likely surrounded by a layer of proteins. This dynamic protein "corona" depends on the concentration in the biological fluid and hence the composition of the layer varies in different parts of the body (Cedervall et al., 2007; Lundqvist et al., 2008; Lynch et al., 2007; Walczyk et al., 2010,). Thus, the reactions in the body to such a NP-protein complex is most likely different from that induced by the bare NP and possibly affecting biodistribution and thereby causing unwanted side effects (Adiseshaiah et al., 2010; Leszczynski 2010,). The function of protein coating is not fully known but since most nanoparticles show strong affinity for proteins, it is of importance to investigate this interaction in different fluids. In blood, plasma proteins constituting the NP corona is possibly affecting a wide range of effects such as phagocytosis via immunoglobulins and complement (Dobrovolskaia & McNeil 2007), coagulation via prothrombin (Dobrovolskaia 2009) and the distribution of lipoproteins (Benderly et al., 2009; Hellstrand et al., 2009;

The availability and toxicity of any substance to a biological organism is determined by both the concentration/dose that the organism is exposed to, as well as the "toxicokinetics" of the substance. These include the uptake, transport, metabolism and sequestration to different compartments by the organism, as well as the elimination of the substance from the biological organism. These parameters are essential since the potential toxicity of substances is dependent upon the specific organs or cell types exposed, which form the substance is in (e.g. bound to serum protein, aggregated, dissolved, oxidized), as well as the period of time the substance interacts/remains at the site of primary and secondary exposure. These parameters are influenced by the physical–chemical characteristics of the substance, therefore a detailed characterization of the substance is pivotal in order to allow

McNeil, 2008).

Zensi et al., 2010).

generalizable conclusions and should therefore be given ample attention. Furthermore, most of these toxicological parameters involve proteins and/or actions carried out by proteins. Thus it is pivotal to the understanding of NP toxicology, and thereby the possibility to predict health effects caused by NPs, to understand NPs interactions with proteins. One of the best, if not the best, technique to separate proteins is two-dimensional gel electrophoresis (2-DE). Preferably, this is combined with peptide mass fingerprinting and MALDI-TOF MS analyses for fast identification of the separated proteins, which then may be followed by tandem MS analyses for sequence information. Here we present our results regarding serum protein interactions with metal-nanoparticles (Al2O3, ZnO/Al6%, SiO2) and Carbon Nanotubes, obtained using 2-DE and MS. Furthermore, we elaborate on and exemplify different aspects of the 2-DE/peptide mass fingerprinting-technique to further improve the approach.
