**2.4 Isolation of lipoproteins**

#### **2.4.1 HDL isolation**

330 Gel Electrophoresis – Advanced Techniques

Commercial SiO2 size 0.007µ (S-3051), ZnO/6% Al doped <50nm(677450), and Al2O3 <50nm (544833) were purchased from Sigma Aldrich. As comparison, single walled Carbon Nanotubes (CNO, 704121) from Sigma Aldrich was used. Nanoparticles, characterized by manufacturer, were dispersed in Milli-Q water and/or PBS (137 mM NaCl, 2.7 mM KCl, 8.45 mM Na2HPO4, 1.47 mM KH2PO4 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 22C. For temperature stabilizing purposes, samples were placed in a thermostat bath (22C) 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 20C (0.911) and 25 C (1.023) (Hackley &

Data analysis was performed using a nonlinear fit model via ALV-Regularized Fit (ALV-Correlator Software Version 3.0. using ALV-Regularized in nonlinear fit model

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. SiO2, ZnO/6% Al doped, Al2O3 and CNO were then exposed to three plasma samples respectively. In all nanoparticle

Nanoparticles were dissolved (final concentration 2 mg/ml) in PBS and incubated with 1% plasma at 37C 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)

**2. Methods** 

Clogston, 2007).

http://www.alvgmbh.de/).

**2.2 Preparation of human plasma** 

exposures fresh plasma was used.

**2.3 Nanoparticle/plasma incubation** 

was applied on the IEF strip in each 2-DE analysis.

**2.1 Characterization of nanoparticles** 

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, 700*g*) 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 290 000*g* 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 (NH4HCO3, 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.

#### **2.4.2 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.

#### **2.5 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 Na2H2PO4, 0.15M NaCl, pH 7.4), 50 µl of plasma

Two-Dimensional Gel Electrophoresis and Mass

**2.8 Isolation of protein spots** 

dehydration with 100% ACN.

further MS preparation, the proteins were stored at -70ºC.

**2.9.1 Tryptic digestion** 

**2.9.2 Glu-C digestion** 

**2.9.3 Cyanobromide digestion** 

**2.9.4 Endoproteinase Asp-N digestion** 

**2.9 Digestion** 

Spectrometry in Studies of Nanoparticle-Protein Interactions 333

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

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 37º 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

Glu-C (Roche, Basel, Switzerland) was diluted with 25 mM NH4HCO3, 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.

One Cynobromide (CNBr, Sigma-Aldrich) crystal was dissolved in 250 µL 70% TFA (in dH2O). 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.

Asp-N (P3303, Sigma-Aldrich) was diluted with 100 mM NH4HCO3 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

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.
