Two-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions

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.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. 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. Here, by using a proteomic approach, we present results showing that several proteins indeed are associated to NPs that have in vitro been introduced to human blood plasma.


Introduction 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).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 (Al 2 O 3 , ZnO/Al6%, SiO 2 ) 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.

General introduction to the 2-DE technique
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 toxicology.
Nanoparticles, characterized by manufacturer, were dispersed in Milli-Q water and/or PBS (137 mM NaCl, 2.7 mM KCl, 8.45 mM Na 2 HPO 4 , 1.47 mM KH 2 PO 4 pH 7.3) and sonicated on ice for 10 min.The hydrodynamic sizes of the particles were analyzed by Dynamic Light Scattering (DLS).DLS measurements were performed using an ALV/DLS/SLS-5022F system (ALV-GmbH, Langen Germany) and a HeNe laser at 632.8 nm with 22 mW output power.The scattering angle was 90 and the temperature 22C.For temperature stabilizing purposes, samples were placed in a thermostat bath (22C) for at least 10 minutes prior to the measurements.Samples were diluted in Milli-Q water or PBS.Ultrasonication of two different kinds was performed to decrease the degree of agglomeration; either an ultrasonic bar homogeniser (Sonoplus HD 2200, Bandelin electronics, Germany) was used or samples were placed in an ultrasonic bath (USC300T, VWR, Sweden).The viscosity of PBS was set to 0.9782 mPa's by linear interpolation between tabulated values for 20C (0.911) and 25 C (1.023) (Hackley & Clogston, 2007).

Preparation of human plasma
Human plasma, collected in sodium citrate tubes, was prepared from three healthy volunteers.After cooling, the blood was centrifuged in 800g for 10 min and plasma, free from red blood cells, was drawn from the top of the tube.SiO 2, ZnO/6% Al doped, Al 2 O 3 and CNO were then exposed to three plasma samples respectively.In all nanoparticle exposures fresh plasma was used.

Nanoparticle/plasma incubation
Nanoparticles were dissolved (final concentration 2 mg/ml) in PBS and incubated with 1% plasma at 37˚C for 1h.As control, to ensure there was no protein precipitation, one sample was prepared without nanoparticles.Unbound proteins were separated from nanoparticles by centrifugation for 40 min at 50 000g and 4°C.The supernatant was discarded and the particle pellet was washed in Dithiothreitol (DTT, Sigma-Aldrich) 20mM/-acetone buffer followed by a second centrifugation step for 10 min at 50 000g and 4°C.The supernatant was discarded and the pellet was air dried.The pellets containing nanoparticles and attached proteins were then dissolved in denaturing solution containing 9M Urea (Sigma-Aldrich), 65mM DTT and 4% (3-(3-cholamidopropyl)-dimethylamino)-1-propanesulfonate (CHAPS).The solution was incubated at room temperature for 30 min before denatured proteins were separated from the nanoparticles by centrifugation for 30 min at 50 000g and 4°C.The supernatant was collected and the samples were analyzed in triplicates.20 µl (40µg protein) was applied on the IEF strip in each 2-DE analysis.

HDL isolation
Preparation of high density lipoprotein (HDL) was performed by a method described by Sattler et al.1994, with slight modifications (Karlsson et al., 2005).Blood samples in EDTAcontaining tubes were obtained from healthy volunteers after an overnight fast.After centrifugation (10 min, 700g) at room temperature, plasma was collected.EDTA (1 mg/mL) and sucrose (final concentration 0.5%) were added to prevent HDL oxidation and aggregation, respectively.Five milliliters of EDTA-plasma adjusted to a density of 1.24 g/mL with solid KBr (0.3816 g/mL) was layered in the bottom of a centrifuge tube (Beckman, Ultraclear tube).The EDTA plasma fraction was gently overlayered with KBr/PBS solution (0.0834 g KBr/mL, total density 1.063 g/mL).In one centrifuge tube, proteins were stained with Coomassie Brilliant Blue to be used as a reference while collecting the HDL fraction.Ultracentrifugation was performed in a Beckman XL-90 equipped with a Ti 70 rotor (fixed angle; Beckman Instruments, Fullerton, CA, USA) for 4 h at 290 000g and 15ºC.By this procedure the lipoprotein fractions with a density lower than 1.063 g/mL (low density and very low density lipoprotein) are located at the top of the tube and HDL is located in the middle of the tube.HDL was collected by penetrating the tube with a syringe.To avoid contamination by serum proteins, HDL were then further purified by a second centrifugation step.KBr/PBS solution (0.3816 g KBr/mL) was added to the HDL (total density 1.24 g/mL) and the centrifugation was performed under the same conditions as described above, but for 2 h.HDL was collected from the top of the tube and desalted using desalting buffer (NH 4 HCO 3 , 12mM, pH 7.1) and PD 10 columns (Sephadex™ G-25 M, GE Healthcare, Buckinghamshire, United Kingdom ).Protein concentration in the HDL solution was determined with Bio-Rad protein assay (Bio-Rad, Richmond, CA, USA).Sample (3.5 mL) was lyophilized and dissolved in 0.25 mL sample solution (9 M Urea, 4% CHAPS, 2% Pharmalyte , 65 mM DTT, 1% bromophenol blue) according to Görg et al.1988.

Immunoaffinity chromatography
Anti-ApoA-I antibodies were attached to a 5 mL HiTrap NHS-activated HP column (GE Healthcare) according to manufacturer's instructions.2.5 mL plasma were desalted by the use of PD-10 columns and the eluted sample were diluted to 4 mL with 50 mM Tris-HCl, 0.15M NaCl, pH 7.5.The ApoA-I coupled immunoaffinity column were equilibrated by allowing 10 column volumes flow through it.Desalted sample were applied into the column and allowed to recirculate for 40 minutes.Loop were disconnected and washed with 10 column volumes of 50 mM Tris-HCl, 0.15M NaCl, pH 7.5 followed by ten column volumes of 50 mM Tris-HCl, 0.5M NaCl, pH 7.5.ApoA-I adsorbed to the column were eluted with 20 mL of 0.1M Glycin-HCl, pH 2.2.Sample was collected in fractions of 0.4 mL in tubes which each contained 20 µL 1M Tris, pH 9.0 for pH-neutralization of the sample.Fractions containing proteins were pooled and desalted using PD-10 columns.

Albumin and IgG removal
The removal of the high abundance proteins albumin and IgG from plasma was performed using an Albumin and IgG removal kit (GE Healthcare).Briefly, the column was equilibrated with binding buffer (20mM Na 2 H 2 PO 4 , 0.15M NaCl, pH 7.4), 50 µl of plasma was diluted to a volume of 100 µl and applied to the column.After 5 min incubation, the depleted sample was collected in an eppendorf tube by centrifugation at 784g.The total protein concentration before/after depletion was determined with Bio-Rad protein assay.After depletion of high abundant proteins the samples were desalted using PD-10 columns.

2-DE analysis
2-DE was performed using IPGphor and Multiphor (GE healthcare).Briefly, the proteins were resuspended in 150 μL of a 2-DE sample buffer containing 9 M urea, 65 mM DTT, 2% Pharmalyte (GE Healthcare), 4% CHAPS, and 1% bromophenol blue and then centrifuged at 4ºC and 23000g for 30 min to remove debris.The supernatant was then mixed with a rehydration buffer consisting of 8 M urea, 4% CHAPS, 0.5% IPG buffer 3-10 NL (GE Healthcare), 19 mM DTT, and 5.5 mM Orange G to a final volume of 350 μL.The first dimension was performed by in-gel rehydration for 12 h in 30 V on 18 cm pH 4-7 linear or pH 3-10 nonlinear IPG strips (Immobiline DryStrips, GE Healthcare).The proteins were then focused at 53000 Vh at a maximum voltage of 8000 V (Görg et al., 2000).The second dimension (SDS-PAGE) was performed by transferring the pI focused proteins (IPG strips) to homogeneous or gradient home-cast gels on gel bonds.The electrophoresis was performed at 40-800 V, 10°C, 20-40 mA, overnight.

Staining and image analysis
Sypro Ruby (Bio-Rad) staining were done according to manufacturer's instructions.In short, gels to be stained with Sypro Ruby were directly placed in a fixing solution containing 10% methanol and 7% acetic acid for at least 20 minutes after 2-DE.Gels were then washed 3x10 minutes under agitation with Milli-Q water before approximately 400 mL of Sypro Ruby stain were added and incubated in room temperature over night.
Silver staining of gels were done according to Shevchenko et al. 1996, with some few modifications.Proteins were fixed by incubating the gel in 50% methanol and 5% acetic acid for at least 20 minutes directly after 2-DE and then incubated with 50 % Methanol for 5 minutes, followed by Milli-Q water for 10 minutes.In the sensitizing step the gel was incubated with 0.02 % sodium thiosulphate for 1 minute, followed by 2x1 minutes washing with Milli-Q water.The gel was then immersed in 0.1 % silver nitrate solution for 20 minutes before excess of silver was washed away by 2x1 minute in Milli-Q water.Next, the gel was developed in 0.04 % formaldehyde in 2 % sodium bicarbonate solution for 2x1 minute.The exact developing time was optimized depending of the protein amount in the gel.Finally, the reaction was stopped by incubation 1x5 min in 0.5 % glycine and the gel washed with Milli-Q water for 2x20 min.
The images of the protein patterns were analyzed by a CCD (Charge-Coupled Device) camera digitizing at 1340*1040 pixel resolution in a UV scanning illumination mode for Sypro Ruby stained gels or at 1024*1024 pixel resolution in white light mode for silver stained gels using a Flour-S-Multi Imager in combination with a computerized imaging 12bit system (PDQuest 2-D gel analysis software, version 7.1.1).The unit of the UV light source is expressed in counts while the unit of the white light source is expressed as optical density (OD).Gel images were evaluated by spot detection, spot intensities and geometric properties.
www.intechopen.comTwo-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions 333

Isolation of protein spots
Protein spots were excised from the gels using a syringe and transferred to eppendorf tubes.For silver destaining, 25 µL of 100 mM sodium thiosulphate and 25 μL of potassium ferricyanide were added to the gel pieces (Gharahdaghi et al., 1999).When the pieces were completely destained, the chemicals were removed by washing (6×5 min with Milli-Q water) before addition of 50 μL of 200 mM ammonium bicarbonate and incubation for 20 min at room temperature.The gel pieces were washed (3x5 min with Milli-Q water) and dehydrated with 100% acetonitrile (ACN) for 5 min or until the gel pieces were opaque white.After removal of the ACN, the gel pieces were dried in a SpeedVac vacuum concentration system (Savant, Farmingdale, NY).Protein spots excised from Sypro Ruby stained gels were washed in 50% ACN/25 mM ammonium bicarbonate 2x30 min prior to dehydration with 100% ACN.

Tryptic digestion
The protein spots were excised from the gel with a syringe and transferred to small eppendorf tubes (0.5 mL).Proteins from fluorescently stained gels were visualized and excised on a blue light transluminator (DR-180 B from Clara Chemical Research, Denver, CO, USA) wearing darkened amber glasses.After destaining and dehydration, about 25 µL trypsin (20 mg/mL in 25 mM ammonium bicarbonate, Promega, Madison, WI, USA) was added to each gel piece.To minimize autocatalytic activity, the samples were kept on ice for 30 min, prior to incubation in 37 º C over night.The supernatant was transferred to a separate tube and the peptides were further extracted from the gel piece by incubation in 50% ACN/5% trifluoroacetic acid (TFA, Sigma-Aldrich) for 5 h at room temperature.The supernatant from the two steps was then pooled and dried in SpeedVac until complete dryness.If not dissolved in 5 µL 0.1% TFA for further MS preparation, the proteins were stored at -70ºC.

Glu-C digestion
Glu-C (Roche, Basel, Switzerland) was diluted with 25 mM NH 4 HCO 3 , pH 7.8, to a concentration of 20 µg/mL.25 µL was added to each gel piece and incubated at room temperature over night.The supernatant was then dried in SpeedVac until complete dryness.If not dissolved in 5 µL 0.1% TFA for further MS preparation the proteins were stored at -70ºC.

Cyanobromide digestion
One Cynobromide (CNBr, Sigma-Aldrich) crystal was dissolved in 250 µL 70% TFA (in dH 2 O). 25 µL CNBr in 70% TFA was added to the dried gel piece and incubated in darkness in room temperature overnight.The supernatant was then dried in SpeedVac until complete dryness.If not dissolved in 5 µL 0.1% TFA for further MS preparation the proteins were stored at -70ºC.

Endoproteinase Asp-N digestion
Asp-N (P3303, Sigma-Aldrich) was diluted with 100 mM NH 4 HCO 3 pH 8.5 to an enzyme concentration of 8 µg/mL.25 µL was added to one tube containing one gel piece.To minimize autocatalytic activity, the samples were kept on ice for 30 min, prior to incubation in 37 º C over night.The supernatant was then dried in SpeedVac until complete dryness.If not dissolved in 5 µL 0.1% TFA for further MS preparation the proteins were stored at -70ºC.
HDL was desalted in a PD-10 column and eluted in a 50 mM Na 2 HPO 4 , pH 6.0 buffer.A volume corresponding to 1200 µg of HDL proteins were then incubated with 10 U of Neuraminidase (Sigma-Aldrich, N3786) in 37˚ C for 4 hours.After incubation, sample was desalted in a PD-10 column with desalting buffer (NH 4 HCO 3 , 12mM, pH 7.1) and subsequently frozen in -70ºC.Sample was lyophilized before 2-DE.

ZipTip
After digestion and drying of peptide samples, some were desalted and purified by the use of ZipTip C18 ® pipette tips (Millipore, Billerica, MA, USA).Samples were diluted up to 10 µL with 0.1% TFA.A ZipTip was wet by loading 3x10 µL with 50% ACN and discarding the liquid.Ziptip was equilibrated by loading 3x10 µL of 0.1% TFA and discarding the fluid before peptide sample was carefully loaded into the ZipTip by pipetting.The ZipTip was washed with 5x10 µL of 0.1% TFA before peptides were eluted with 10 µL 50% ACN.

Mass spectrometry
Peptides obtained after digestion were mixed 1:1 with matrix, -Cyano-4-hydroxycinnamic acid (CHCA, 0.02 mg/mL) or 2,5-dihydroxybenzoic acid (DHB, 0.02 mg/mL) in 70% ACN/0.3%TFA, and then spotted onto a stainless steel target plate.Analyses of peptide masses were performed using MALDI TOF-MS (Voyager DE PRO; Applied Biosystems) equipped with a 337 nm N2 laser operated in reflector mode with delayed extraction.Positive ionization, a delay time of 200 ns, and an accelerating voltage of 20 kV were used to collect spectra in the mass range of 600-3600 Da.Data processing of the spectra was performed in a Data Explorer TM Version 4.0 (Applied Biosystems).External mass calibration with a standard peptide mixture and internal calibration using known trypsin autolysis peaks (m/z 842.5100, 1045.5642, 2211.1046) were also performed prior to the database search.For tandem MS analysis, the digested peptides were dried and dissolved in 10 µL 0.1% TFA.The peptides were desalted and purified by using ZipTip C18 ® columns.Elution was acidified by the addition of 1% formic acid.About 2 µL of the sample was applied into a silver-coated glass capillary and analyzed by a hybrid (triple quadrupole-TOF) mass spectrometer (API Q-STAR Pulzer; Applied Biosystems) equipped with a nanoelectrospray ion source (MDS-Protana, Odense, Danmark) operated in the nanopositive mode.Data processing was performed with Analyst QS software (Applied Biosystems).Fragmentation spectra were interpreted manually.

Database search
Peptide masses (major peaks) in the spectra were submitted to database search.NCBI and Swiss-Prot were used with Aldente or MS-Fit as search engines.Restrictions were human www.intechopen.comTwo-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions 335 species, mass tolerance >75 ppm in most of the searches, maximum one missed cleavage, and cysteine modification by carbamidomethylation. MS-Digest, MS product, and BLAST search was used for protein identification of the derived tags resulting from amino acid sequencing with MS/MS.In peptide mass fingerprinting, protein matches with p-values below 0.05 are used and with LC-MS/MS analyses an FDR < 1 % is considered significant.

Western blot
Plasma proteins were separated on 2-DE.Proteins were then transferred to a 0.2 μm PVDF membrane.After blocking with 5% non-fat dry milk in Tris buffered saline (TBS) overnight, the membrane was washed two times with Tween-Tris buffered saline (TTBS, pH 7.5) and then incubated overnight with primary rabbit anti human C-III antibodies (Abcam, 21032, 1:5000) in 2% non-fat dry milk in TTBS (pH 7.5) at room temperature.After washing four times with TTBS, the membrane was further incubated for 1h with secondary goat anti rabbit antibodies conjugated with horse radish peroxidase (HRP, 170-6515, BioRad,1:40 000) in 2% non-fat dry milk in TTBS (pH 7.5) at room temperature.In order to visualize the proteins the PVDF membrane was treated with ECL Plus Western Blotting Detection System (GE Healthcare) and then exposed to X-ray film (AGFA Medical, Mortsel, Belgium).

Nanoparticle characterization
DLS, which is also known as Photon Correlation Spectroscopy or Quasi-Electron Light Scattering, is a technique used to study the size and size distribution of particles suspended in a liquid.The technique is based on the scattering of light of particles in diffusive random (brownian) motion.The average displacement for the Brownian motion is defined by the translational diffusion coefficient (D).The particle diffusive motion in liquid is size dependent, and a larger particle has a slower motion as compared to a smaller particle.This brownian motion can be investigated by irradiating the sample with a coherent laser and studying the intensity fluctuations of the scattered light (Finsy, 1994).
Particle sizing can be done in several ways.Typically the information retrieved from different techniques is to some extent diverse, as each technique is sensitive to it´s specific properties of the particles.That means that a technique which is based upon the scattering intensity does not deliver the same size or size distribution as a technique that is based upon the projected area or the density of a nanoparticle.For nano sized particles, transmission electron microscopy (TEM) is frequently used in purpose to study the size and the shape of particles.In TEM, the sample preparation together with the measurement is relatively time consuming and furthermore the measurement is limited only to a very small fraction of the sample.This means that a lot of replicates must be studied in order to achieve good statistics.A dynamic light scattering measurement on the other hand, is fast and convenient as it usually takes only a few minutes to perform.Data recording procedure is thus short but the analysis and interpretation requires knowledge and care.DLS measurements must be performed with highly diluted solutions, to avoid multiple scattering phenomenons and misleading artifacts are frequently present in DLS studies.
Particle sizes obtained when measuring with DLS are by default larger than those obtained when analyzing the material with TEM.The size calculated from the translational diffusion coefficient in DLS generally is referred to as the hydrodynamic diameter, e.g the diameter of a sphere having the same diffusion coefficient as the particle, while the size of the nanoparticles obtained from TEM is the core size of the nanoparticle investigated.It should be noted that in many cases when the sample consists of a mixture of nanoparticles with a range of sizes and/or mixture of particle shapes, results should be taken as an estimation only but clearly trends can be observed.
The autocorrelation function of the scattered intensity results in an average value of the product of the intensity at time t and the intensity at a time delay later t+dt.The value obtained from the correlation function is large for short delays, since the intensities are highly correlated.The value will be low for longer delays; i.e. the autocorrelation can be described as a decaying function of time delay.From the autocorrelation function, the diffusion constant D can be determined.Furthermore, by using Stokes-Einstein equation the corresponding size distribution is calculated according to: where k B is the Boltzman constant, T the temperature, η the viscosity of the solvent and d the hydrodynamic diameter of the particles.This implies that the temperature must be constant during the measurement and the viscosity of the sample solvent must be known.It should be noted that the formula shown above (Equation 1) is valid only for non-interacting spherically shaped particles, i.e experimental data are fitted to a model assuming spherical particles (Finsy, 1994).
Figure 1 shows five different functions representing the typical information retrieved in DLS in the actual measurement and by using algorithms.The measured data in DLS is the correlation function (Figure 1A).This function holds information about the diffusion of particles in the sample and can be transformed to a graph showing the decay time of light scattering fluctuations (Figure 1B).From the decay time distribution function, the values of the particle radius (Figure 1C) can be calculated using the Stokes-Einstein equation.
The amount of light that is scattered from a particle is dependent on the particle size.
According to the Rayleigh theory (Sorensen, 2008), the scattering factor roughly is proportional to the sixth power of the particle size.This means that a small particle scatter light less than a larger particle and thus different weights have to be applied to transform the intensity weighted data to a useful size distribution.These weights can be mass based (Figure 1D) or number based (Figure 1E).Powder based samples that are dispersed in a liquid often are severely aggregated.Ultrasonic baths can be used to decrease the aggregation in the solution.Choice of solvent is also important and it clearly affects the capability of dispersing nanoparticles.
A Figure 3 shows the size distribution of Aluminium doped ZnO nanoparticles dispersed in water and PBS.Bar ultrasonication introduces smaller sized populations, in consistence with the results in Figure 2. The hydrodynamic radii of these particles in MilliQ water are around 30 nm as largest according to Figure 3.This could be compared with the information from the supplier that these particles are < 50 nm as measured by TEM.Again hydrodynamic diameter is always larger that the core size of nanocrystals obtained from TEM.In this case the sample is also aggregated in solution, which produces even larger sizes and size distributions.
The SiO 2 particles are 7 nm sized as primary particles according to the supplier based on calculations using the surface area as measured by the nitrogen adsorption method of Brunauer (Brunauer et al., 1938).The supplier also states that these particles commonly form  In conclusion, it could be said that size and size distribution of nanoparticle samples could be estimated by DLS.Valuable information as the trends in size distribution connected to sample preparation methods and choice of solvent can be obtained.Sample preparation methods are indeed very important as well as choice of solvent.Care should be taken when choosing fitting model and the model-inbuilt parameters.Inhomogeneous samples are less straight forward to analyze.Presence of aggregates is easily detected.In summary information obtained from DLS is important for everybody that is doing research on nanoparticles in liquids.The numbers given as product information i.e. the size and size distribution, are often relevant for the core-size of the nanocrystals within the material.However the nanoparticles are most often not soluble to that extent.Nanoparticles obtained in dry state and then dispersed in liquid usually form aggregates as shown in this study.

Nanoparticle-plasmaprotein interactions
In a previous study performed by us, the inflammatory response in human monocyte derived macrophages after exposure to wear particles generated from the interface of studded tires and granite containing pavement (Karlsson et al. 2011) was investigated.Particle characterization showed that dominating peaks in the EDX spectra were Silica and Aluminium.Particles of nanosize were also present (SMPS), but it was not possible to characterize them due to low abundance.As a result of their very small diameter (< 0.1 µm), inhaled nanoparticles are believed to be predominantly agglomerated and deposited in the periphery of the lungs, where they interact with cells such as macrophages and epithelial cells (Beck-Speier et al. 2005) but they may also translocate into the circulation, which is a critical step, since their fate in vivo in not known.Investigating plasma protein-nanoparticle interactions with a toxico-proteomic approach is a useful tool to improve our knowledge about the effects of nanoparticles of different origin, size and surface properties in biological systems.
In purpose to mimic a potential exposure to airborn nanoparticles translocated into the circulation, commercial SiO 2 and Al 2 O 3 were mixed with plasma proteins.As comparison, commercial ZnO (Al-doped 6%) and a non-metal oxide; single walled Carbon Nanotubes was used.All preparations were performed in triplicates with three different subjects exposed to each type of particles.The protein patterns resulting from the three different exposures of commercial SiO 2 , Al 2 O 3, ZnO and CNO respectively were identical.
Particle characterization and estimation of particle agglomeration prior to exposure is crucial.In a recent study of nanoparticle-plasma protein interactions (Deng et al. 2009), the DLS spectra indicated large agglomerates prior to plasma protein exposure.Most likely, and in line with the authors suggestions (Deng et al. 2009), complexes with hydrodynamic size of 10000-100 000 nm do not result in the same protein patterns as the interactions of smaller particles/agglomerates and plasma proteins.In our optimized protocol, with different particle origin, less gentle bar sonication instead of in water bath and thereby reduced hydro dynamic sizes of the agglomerates (Fig 2-4) -an altered pattern of interacting proteins was indeed found (Figure 5, Table 1).
Interestingly, the interaction of SiO 2 and CNO with plasma proteins resulted in very similar protein patterns despite their different properties (Fig 5).It has to be stated though, that the fate of CNO in the lung may not be translocation into the circulation due to the tube like structure, but CNO is also of interest for medical applications (Wu et al. 2011).
The transport proteins Albumin and Alpha-2-HS-glycoprotein interacted with SiO 2, CNO and ZnO but not Al 2 O 3 while Transferrin interacted with SiO 2, Al 2 O 3 and notably also CNO but not ZnO.Supporting our results, the binding of albumin to single walled CNO has previously been described to promote uptake by the scavenger receptor in RAW cells (Dutta et al., 2007).In line, intravenous administration of CNO has in a different study resulted in high localization in the liver (Cherukuri et al., 2006).Another possible way for nanoparticles into the cells, are as Transferrin/particle complexes that are able to enter the cells via the Transferrin receptor.The Transferrin receptor is an interesting and relevant target in cancer research since its expression is increased in tumor cells.In a recent study, Transferrin covalently attached to silica nanoparticles carrying a hydrophobic drug caused an increase in mortality of the targeted cancer cells compared to cells exposed to nontargeted particles and www.intechopen.comTwo-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions 341 free drug (Ferris et al. 2011).What demand further studies though, is the fate of the silica particles in a longer perspective, taken up by tumor cells as well as other cells.A third transport protein, the thyroxin transporting protein Transthyretin, also known to bind toxic components in the blood stream (Hamers et al., 2011), was found to interact with CNO and SiO 2. This finding confirms a previous study that pointed out that silica interaction (inhaled) with plasma Transthyretin is contributing to the stabilization of fibroids in rat lungs (Kim et al., 2005).The possible effects of CNO interaction with Transthyretin remains to be investigated.Fig. 5. Plasma protein binding profiles of different nanoparticles.Four different nanoparticles; Al 2 O 3 , CNO, ZnO (* Aluminium-doped 6%) and SiO 2 , were mixed with human plasma and isolated through ultracentrifugation.The protein contents were then separated by 2-DE and silver stained.Bound proteins were identified by MS as shown in Table 1.
The inflammatory marker Fibrinogen was found to interact with SiO 2, Al 2 O 3 and CNO but interestingly not with ZnO (Fig 5).Fibrinogen binds foreign substances in the circulation and promotes macrophage activation -a mechanism that may result in retention of particle/protein complexes in the intima with accompanying cardiovascular complications (Shulz et al., 2005).On the other hand, Lysozyme C, a well known anti-bacterial agent, was only found on Al 2 O 3 and ZnO.Under the present condition, it was unfortunately not determined if the binding to ZnO occurred due to the 6% Al doping of the ZnO.Lysozyme has previously been found to interact with nano-TiO 2 particles (Xu et al., 2010).They reported that the coexistence of nano-TiO 2 particles and Lysozyme resulted in the transition of Lysozyme conformation from -helix into -sheet secondary structure and a substantial inactivation of Lysozyme.Moreover the -sheets are able to induce the formation of amyloid fibrils, a process which plays a major role in pathology.

Number in
Figure 2  Furthermore, some antigen binding proteins; IgKC, DKFZ and IgLC, were also found to interact with Al 2 O 3, CNO and SiO 2 but were not detectable on ZnO (Fig 5).Immunoglobulins are able to activate the complement system but they also often represent unspecific binding during protein purification caused by insufficient washing.Our results compared to previous findings indicates that increased hydrodynamic size of nanoparticle agglomerates seems to correlate to increased amounts of immunoglobulins.Overall ZnO was not binding as many proteins as the other particles and may even bind less without being Al-doped but an interesting finding in the ZnO preparation was a protein only described on transcript level and highly similar to the protein Amyloid A4 precursor.This family of proteins acts as chelators of metal ions such as iron and zinc.They are also able to induce histidinebridging between beta-amyloid molecules resulting in beta-amyloid-metal aggregates and it has been reported that extracellular zinc-binding increases binding of heparin to Amyloid A4 (Uniprot 2011).
The protease inhibitor Alpha-1-antitrypsin, was found in the CNO, SiO 2 and ZnO preparations but not in Al 2 O 3 while another antioxidant, Haptoglobin was found only on CNO and SiO 2. To our knowledge, the binding of Alpha-1-antitrypsin to the nanoparticles in www.intechopen.comTwo-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions 343 this study has not been described previously but an increase of Haptoglobin has been reported in a study investigating acute phase proteins as biomarkers for predicting the exposure and toxicity of nanomaterials (Higashisaka et al., 2011).
At last, the HDL associated Apo A-I, with well known anti-endotoxin activity (Henning et al., 2006) and receptor interaction properties was found in all preparations but was most abundant after SiO 2 exposure .Apo A-I may be acting as a scavenger clearing the particles from the blood stream via the scavenger class B-I receptor (SRBI).The SRBI receptor is mainly located on the liver and plays an important role in cholesterol efflux (Verger et al., 2011) but is also present on other cells (Mooberry et al. 2010).Apolipoproteins in general are of interest for the pharmaceutical industry as carriers of nanoparticle bound drugs for brain uptake (Kreuter et al., 2005) since apo E and apo B-100 are taken up by the cells via receptor mediated endocytosis.The hypothesis is that the nano-particle/apolipoprotein complex mimics the natural lipoprotein particle.The identity of Apo A-I was, as the other proteins, confirmed by peptide mass fingerprinting using MALDI TOF MS (Table 1) and one dominating Apo A-I peptide in the MS spectra (Figure 6A) was in addition sequenced by MS/MS (Figure 6B) to further confirm the identity.Overall the binding of plasma proteins to nanoparticles, based on previous and our findings, seems to vary with origin, surface properties, size and thereby also diameter of agglomerates.Particle characterization prior to exposure for plasma proteins or cells is therefore extremely important to receive reliable results that are possible to interpret.
Interacting proteins under the present conditions are dominated by proteins involved in the immune defense and reverse transport to the liver but notably also proteins mediating brain uptake.

Plasma sample preparations
Sample preparation is an important step that influences the separation of proteins with 2-DE.A common problem in most biological samples is the presence of salt ions.In 2-DE, salt concentrations >10 mM affects the isoelectric focusing step and markedly reduces the effectiveness of the charge separation.There are several easy ways to remove salts, e.g. by precipitation of the proteins or by gelfiltration in small desalting columns.Plasma samples are possible to analyze directly with 2-DE since the high protein concentration allows a simple dilution of the sample to lower the salt concentration.However, as illustrated in figure 7, a desalting step besides the dilution still improves the protein pattern.Another well-known problem with biological fluids is the presence of a few highly abundant proteins that may prevail over the low abundant proteins.In plasma, albumin and immunoglobulin G (IgG) constitutes a very large proportion (about 75%) of the total protein loaded on the gels.This may lead to the proportion of low abundant proteins being below the detection limit.Also, the staining of the abundant proteins may interfere with proteins with similar molecular mass and pI.It is therefore advisable to remove albumin and IgG and there are several commercial removal kits available, usually based on antibodies directed towards albumin and IgG.As shown in figure 7, such sample preparation step removes a large fraction of these proteins and increases the proportion of the other proteins in the sample.However, it is important to realize that this step also introduces unspecific removal of proteins and it is our experience that this unwanted loss of proteins varies considerable between the different available removal kits.Plasma contains a wide variety of proteins, many of which are not detectable with 2-DE without further fractionation.In view of the lipid metabolism, important sub-fractions of plasma to study are the lipoproteins.As shown in table 1, one protein that interacts with www.intechopen.comTwo-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions 345 nanoparticles is apo A-I, the major constituent of HDL.This implicates the need of more investigations of HDL as a possible target of nanoparticles that may influence the cholesterol metabolism and increase the risk of cardiovascular disease.HDL can be isolated based on density, size or protein content (e.g.apo A-I) using ultracentrifugation, size-exclusion chromatography or immune-affinity chromatography, respectively, each technique with its own merits and drawbacks.Thus, the rather harsh conditions during ultracentrifugation in high salt gradients may remove weakly associated proteins while the rather mild conditions during chromatography may favor unspecific co-purification of proteins with the cholesterol particle.We have previously mapped the protein content of HDL isolated by two-step density gradient ultracentrifugation (Karlsson et al., 2005).In this study we have compared ultracentrifugation and anti-apo A-I affinity chromatography to isolate HDL from the same plasma sample.As shown in figure 8, more proteins were obviously identified in immune-affinity purified HDL.However, some of the proteins must be considered as possible plasma contaminants as they were not, as e.g. the apolipoproteins, enriched in the HDL fraction.
Fig. 8. Subfractionation of plasma with regard to HDL Plasma (left), HDL purified according to apo A-I with immunoaffinity chromatography (middle) and HDL isolated according to density with ultracentrifugation (right) were separated with 2-DE and silver stained.Proteins were identified with mass spectrometry.

Protein detection
Gel-separated proteins can be visualized by several commonly used staining methods, including dyes (e.g.Coomassie Brilliant Blue and colloidal Coomassie), metals (e.g.silver staining) and fluorescent probes (e.g.Sypro staining and Cy-dyes) (Rabilloud, 2000).The stains interact differently with the proteins and have different limitations with regard to sensitivity, linear range, compatibility with mass spectrometry and type of proteins that stain best.In general, for staining of complex protein samples, silver staining can be considered the most sensitive technique (1-5 ng protein) and Coomassie Brilliant blue the least (50-100 ng) while the sensitivity of colloidal coomassie and the fluorescent dyes are in between.However, it is important to bear in mind that the different stains interact differently with the proteins and therefore one protein may stain very well with one staining method but not with the other (Fig. 9).For example, silver ions react with negatively charged groups and therefore stain glycoproteins containing negative sialic acid very well.
One the contrary, Sypro ruby that binds to proteins through hydrophobic interactions stains hydrophilic glycoproteins quite poorly.Being most sensitive, silver staining is obviously very useful for proteomic approaches.However, the advantage with silver is hampered by its rather low linear range, making it less suitable for quantification than the other staining techniques.For plasma analyses we have therefore adopted a double staining strategy.As illustrated in figure 9, the 2-DE gel is first stained with Sypro Ruby and the proteins are quantified within a high dynamic range.The gel then can be destained and restained by silver to detect additional proteins.As these additional proteins are less abundant their intensities usually are within the limited linear range of the silver staining technique.The proteins may then be selected for MS analyses.As the sample preparation protocol is more time-consuming for silver stained gels than Sypro stained gels it is convenient to pick as many proteins as possible after the first staining step.In this step it is also important to check the optimal destaining time before MS analyses, with plasma samples at least 90 minutes (fig 9).If the samples are not fully destained the signal to noise in the spectra are reduced.

Protein identification with peptide mass fingerprinting using MALDI-TOF MS
Peptide mass fingerprinting with MALDI-TOF MS is an excellent and robust technique for fast identification of plasma proteins after 2-DE (Lahm & Langen, 2000).However, several peptide peaks needs to be detected in the spectra with a high mass accuracy to avoid false www.intechopen.comTwo-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions 347 positive results.There are several approaches to consider for improving the data from the analyses, such as digestion protocol, purification of peptides and choice of matrix.

Alternative digestion of samples
One of the most widely used ways to digest proteins before MS analyses is by trypsin, which cleaves C-terminally of lysine and arginine (not followed by proline).Although lysine and arginine often are distributed in the protein sequences in a way that provides sufficient number of peptides for identification after trypsin cleavage, this is not always so.Furthermore, it is sometimes necessary to use alternative digestion protocols in order to find specific peptides to e.g.characterize differences between protein isoforms.In these cases alternative enzymes, e.g.Asp-N (cleaves N-terminally of aspartic acid and cystein), Glu-C (C-terminally of glutamic acid) or chemical induced cleavage by CNBr that hydrolyzes Cterminally of methionine, is needed.In this study we used Asp-N as a complement to trypsin when identifying the plasma protein serum amyloid A4 (SAA4).This combined digestion approach generated almost 95 % sequence coverage of the protein (figure 10).SAA4 is a constitutively expressed protein which can be found in HDL as differently charged isoforms (fig 14, Karlsson et al., 2005).One explanation to these isoforms could be a small truncation of SAA4 in which one lysine and one tyrosine is removed C-terminally and thereby making the protein more acidic (Farwig et al., 2005).However, by using Asp-N we were able to detect both the intact C-terminal and the intact N-terminal peptide that were not possible after trypsin digestion, ruling out the presence of truncated SAA4 in our sample (figure 10).Besides the use of Asp-N to study SAA4, we have also used Glu-C to analyze apolipoprotein A-I (figure 6) and CNBr to study serum amyloid A-1/2 isoforms (figure 13).Sequence coverage obtained through peptide mass fingerprinting with MALDI-TOF mass spectrometry by the use of trypsin and endoproteinase Asp-N as indicated by the lines.Sequence coverage (without the signal peptide in position 1-18) was 79.5 % with trypsin and 57.1 % with Asp-N.The combined sequence coverage was 93.8%.

Peptide sample cleaning
Peptide samples after in-gel digestion can be cleaned by adsorption to C-18 containing pipette tips (ZipTip®).This clean-up procedure done manually is rather time-consuming but is absolutely necessary before electrospray-quadrupole MS.On the other hand, with a MALDI-TOF instrument, being more insensitive to salts and other contaminants, it is not that obvious.To investigate the possible advantage with ziptip cleaning before MALDI-TOF MS we picked 17 sypro stained proteins after 2-DE.The proteins were digested with trypsin and an aliquot of the obtained peptide solution was purified by ZipTip® (50% ACN elution solution according to the protocol recommended by the supplier), mixed with the matrix CHCA and spotted on the MALDI-target plate and another aliquot of the peptide solution was mixed directly with the matrix and spotted on the same plate.All samples were then analyzed with MALDI-TOF MS with the same settings and with the laser induced collection of spectra in an automatic mode.The spectra were then used for NCBI database search with MS-fit using the same settings for all samples.All 17 proteins were identified with peptide mass fingerprinting in both ziptip cleaned and untreated samples.As shown in Table 2, ziptip cleaning significantly improved the peak intensities, signal to noise ratio and the mass accuracy.This illustrates that removal of salts and other contaminants that will compete with the peptides in the spectra increases the intensities of the peptide peaks and thereby increases the accuracy of the mass determinations and, as a consequence, also increases the reliability of the identifications.On the other hand, the number of peptides and sequence coverage found in ziptip cleaned samples were about the same as in the untreated samples (Table 2).In general, there was a clear tendency that in ziptip cleaned samples more peptides were detected in the lower mass region (<1000 m/z) while fewer peptides were detected in the higher mass region (>2000 m/z).This suggests that the removal of salt ions and low molecular chemicals with subsequent improved signal to noise increases the possibility to detect low molecular mass peptides but that this beneficial effect is counteracted by adsorption of larger peptides to the solid phase of the ziptip.To test this, 10 protein samples were sequential eluted from the ziptip with increasing ACN concentration, up to 90 %.Indeed, this procedure increased the number of peptides found and the sequence coverage increased from 52 +/-16 % in the untreated samples to 58 +/-16 in the ziptip cleaned samples (p<0.05).The effect varied among the different proteins but was in some samples quite profound, almost 2 times higher sequence coverage.It can be concluded from these experiments that purification of peptide samples with ziptip improves the results with MALDI-TOF MS.However, when it comes to the identification of proteins with peptide mass fingerprinting the beneficial effect of the cleaning procedure is quite limited as the number of peptides found, using the standard protocol, is not increased.Therefore, considering the work-load needed for the ziptip procedure, it is doubtful if it is practical to routinely clean samples with ziptip before MALDI-TOF analyses.However, for selected, low abundant, samples it can most likely make a significant difference for the identification.In these cases, sequential elution of the peptides from the ziptip with increasing acetonitrile concentrations is recommended.Transthyretin was identified with sequence coverage of 39 % with CHCA (A) versus 72 % with DHB (B).Peptide peaks marked with an asterisk were matched to the theoretical masses with an accuracy <50 ppm.
Serum amyloid A1 and A2 (SAA1 and SAA2, respectively) are two acute phase proteins, which share more than 95 % sequence identity.Both SAA1 and SAA2 can also be expressed as an alpha-and a beta-form, which are discriminated from the others only in one amino acid position (Strachan et al., 1989).SAA1 and SAA2 are associated to HDL (Karlsson et al., 2005) and are heavily induced by endotoxins (Levels et al., 2011), which is highly relevant in particle toxicology to discriminate between different environmental agents (Karlsson et al. 2011).Based on differences in isoelectric points we were able to separate four isoforms of SAA1/2 in HDL (figure 13A).By peptide mass fingerprinting after trypsin digestion we identified two of the isoforms as SAA1 with pI 5.5 and 6 and two as SAA2 with pI 7 and 8 (figures 13A and 13B).
The theoretical pI of SAA1 and SAA2 is 5.9 and 8.3, respectively.Thus, the pI of one of the isoforms of SAA1 and of SAA2 corresponded to the theoretical values while the other two had an acidic shift (pI 6→5.5 in SAA1 and pI 8→7 in SAA2).N-terminal truncations of SAA1 and SAA2 that would produce such acidic shifts have previously been described (Ducret et al., 1996) and we therefore focused the MS analyses on the N-terminal peptide.As SAA1 and SAA2 contain arginine at the N-terminus we used CNBr, which cleaves before methionines, as an alternative digestion agent to detect the full length N-terminal peptide.These analyses showed that the more acidic isoforms of SAA1 and SAA2 comprised a mixture of truncations with the loss of one, two or four amino acids N-terminally; des-Arg, des-Arg-Ser and des-Arg-Ser-Phe-Phe, respectively, with the loss of arginine being the main explanation to the acidic shifts (figure 13D).On the other hand, the native peptide was only found in the more basic isoforms of SAA1 and SAA2.In total, four forms of SAA1 and four forms of SAA2 was identified.Interestingly, studies of these small molecular mass variants of SAA1 and SAA2 with SELDI-TOF MS indicates population cluster differences in HDL related to the truncations in response to endotoxin (Levels et al., 2011).2-DE makes it possible to separate proteins based on the degree of glycosylation.Hydrophilic sugars affect the binding of SDS and usually render the proteins an apparent higher molecular mass in the second dimension and the presence of negative sialyl-groups makes the proteins more acidic in the first dimension.We have therefore adapted two simple 2-DE mobility shift assays to demonstrate glycosylation of proteins and applied these to study glycosylated isoforms of plasma proteins in HDL.In the first we use endoglycosidase PNGase to cleave N-linked oligosaccharides from the protein backbone and the second is based on enzymatic removal of sialic acid with neuraminidase.As shown in figure 14A, SAA4 is usually expressed in HDL as 6 isoforms, three with molecular masses about 18k and three with molecular masses about 11k.After PNGase treatment it was clearly shown that the 18k isoforms are depending on N-linked glycosylation (Fig 14A).Another glycosylated protein in HDL is apo C-III that can be found as three isoforms; one di-sialylated, one mono-sialylated and one minor non-sialylated form (Bruneel et al., 2008).This was demonstrated by treatment with neuraminidase, which induced a mobility shift with the loss of the two sialylated isoforms and a substantial increase of the non-sialyated apo C-III form (figure 14B).

Conclusions and future research
Overall the binding of plasma proteins to nanoparticles, based on our findings, seems to vary with origin, surface properties and size of the particles.A large portion of the interacting proteins we identified by 2-DE/MS are proteins involved in the immune defense and reverse cholesterol transport to the liver, but we also identified proteins mediating brain uptake.Most likely these protein patterns of the nanoparticles represent a mixture of particle-protein and protein-protein interactions.Extensive research in this field is therefore needed before conclusions could be drawn regarding potential health effects of nanoparticles and their associated protein "corona".One major difficulty to overcome is how to characterize the particles used in different studies in such a way that comparisons www.intechopen.comTwo-Dimensional Gel Electrophoresis and Mass Spectrometry in Studies of Nanoparticle-Protein Interactions 353 and generalized conclusions are allowed.Most types of nanoparticles seem to form aggregates, especially so in water suspensions and the "corona" seen might be heavily influenced by the size/diameter of these aggregates rather than by other particle characteristics.Thus, characterization of particles and the aggregates they form prior to exposure of plasma proteins, cells or other biological systems is therefore extremely important.One way of doing that, as we have showed herein, is by DLS.These analyses gives valuable information about the trends in size distribution connected to sample preparation methods and choice of solvent.Sample preparation methods are indeed very important as well as choice of solvent and care should be taken when choosing fitting model and the model-inbuilt parameters.Thus, information obtained from DLS is important for everybody that is doing research on nanoparticles in liquids.The numbers given as product information i.e. the size and size distribution are often relevant for the core-size of the nanocrystals within the material.However the nanoparticles are most often not soluble to that extent.Consequently, nanoparticles obtained in dry state and then dispersed in liquid usually form aggregates as shown in this study.
Given ample attention to the characterization of the NPs used, future studies of the NPprotein complex behavior in different biological systems are needed.Questions that need to be addressed are which properties of the NPs that govern the protein "corona" formed around the NPs in biological fluids and how these complexes interact with endothelial cells, platelets, cells of the immune system etc.One interesting finding in this study is Amyloid A4, not previously identified in plasma, which was only associated to ZnO particles.This protein may act as a chelator forming metal-amyloid aggregates and needs further attention in toxicological studies.
In summary, the improved 2-DE/MS protocols shown herein underline this proteomic approach as a powerful tool in human nano-particle toxicology.Furthermore, thorough characterisation of the particles studied, e.g. with DLS, is crucial to evaluate the results.

References
Fig. 1.Example of the typical information retrieved in a DLS measurement.The measurement is performed on Al doped ZnO nanoparticles dissolved in MilliQ water.A) shows the correlation function which has a high correlation (close to 1) for really short lag times but decays to zero with a rate dependent on the particle size distribution in the sample.B) shows the normalized distribution function of the Decay time.C) shows the normalized distribution of the unweighted radius and the corresponding normalized distributions of the mass weighted radius and the number weighted radius are shown in D) and E) respectively.

Fig. 2 .
Fig. 2. DLS measurements of Al 2 O 3 nanoparticles.The normalized distribution of the number weighted radii of particles in Al 2 O 3 nanopowder diluted to different concentrations in A) MilliQ and B) PBS as measured by DLS.As marked in the figure, two different kinds of ultrasonic treatment were used to decrease the aggregation before performing the measurement; either an ultrasonic bath or a bar homogenizer.

Fig. 3 .
Fig. 3. DLS measurements of ZnO (Aluminium 6% doped) nanoparticles.The normalized distribution of the number weighted radii of particles in ZnO (Aluminium 6% doped) nanopowder diluted to different concentrations in A) MilliQ and B) PBS as measured by DLS.As marked in the figure, two different kinds of ultrasonic treatment were used to decrease the aggregation before performing the measurement; either an ultrasonic bath or a bar homogenizer.

Fig. 6 .
Fig. 6.Identification of nanoparticle bound apoliprotein A-I with mass spectrometry after 2-DE.A: Peptide mass fingerprint spectrum obtained by MALDI-TOF mass spectrometry after endoproteinase GluC digestion.Asterisks represent peaks corresponding to peptide masses of apo A-I.B: Sequencing of a triply charged peptide (m/z 409.9) corresponding to position 194-203 of apo A-I by collision induced disassociation (CID) in a tandem mass spectrometer.The amino acid sequence with ions corresponding to the different fragments is shown in the upper right corner.

Fig. 7 .
Fig. 7. Preparation of plasma for Untreated plasma (left), desalted plasma (middle) and desalted plasma after removal of albumin and immunoglobulin G (IgG), (right), were separated with 2-DE and silver stained.Arrows indicate protein or protein clusters with increased abundance after treatment compared to untreated plasma.The rectangle indicate an area with improved resolution after desalting.Positions for albumin and IgG chains are indicated with rings.

Fig. 9 .
Fig. 9. Double staining of proteins Plasma proteins were separated with 2-DE and first stained with Sypro Ruby.The gel was then destained with 25 mM ammonium bicarbonate/50 % acetonitrile buffer and the efficiency of the process checked after different time intervals.Finally, the proteins were restained with silver.A: Proteins more stained by Sypro compared to silver.B: Glycoproteins more stained by silver.The rectangle shows an area containing additional proteins detected by silver.Residual Sypro staining of proteins after destaining are indicated with rings.

Fig. 12 .
Fig. 12.Comparison of -cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) as matrices in MALDI-TOF MS of a silver stained protein.Transthyretin was identified with sequence coverage of 39 % with CHCA (A) versus 72 % with DHB (B).Peptide peaks marked with an asterisk were matched to the theoretical masses with an accuracy <50 ppm.

Fig. 13 .
Fig. 13.Identification of serum amyloid A isoforms by 2-DE and MALDI-TOF MS.A: HDL proteins were separated by 2-DE and stained by Sypro Ruby.Arrows indicate the two isoforms of serum amyloid A1 (SAA1) and the two isoforms of serum amyloid A2 (SAA2) identified by peptide mass fingerprinting.B and C: MS spectra after trypsin digestion of SAA1and SAA2, respectively, with specific masses indicated.D: MS spectra after CNBr digestion of SAA1.Masses corresponding peptides from N-terminal truncated variants of the protein (protein spot 1) and the mass corresponding to the N-terminal peptide of the native protein (protein spot 2) are indicated.

Fig. 14
Fig. 14. 2-DE mass and charge mobility shift assays to demonstrate glycosylated protein isoforms.A: SAA4 analyzed by 2-DE and silver stained.N-linked glycosylated serum amyloid A4 (SAA4) isoforms shown by deglycosylation with PNGase.B: Apo C-III analyzed by 2-DE and Western blots.Sialylated apo C-III isoforms shown by desialylation with neuraminidase.