**1.2 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.

Two-Dimensional Gel Electrophoresis and Mass

**2.4.2 Immunoaffinity chromatography** 

**2.5 Albumin and IgG removal** 

**2.4 Isolation of lipoproteins** 

**2.4.1 HDL isolation** 

Spectrometry in Studies of Nanoparticle-Protein Interactions 331

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.

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

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

containing proteins were pooled and desalted using PD-10 columns.
