**2.9.2 Glu-C digestion**

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.

#### **2.9.3 Cyanobromide digestion**

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.

#### **2.9.4 Endoproteinase Asp-N digestion**

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

Two-Dimensional Gel Electrophoresis and Mass

**2.13 Western blot** 

**3. Results and discussion** 

**3.1 Nanoparticle characterization** 

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.

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).

studying the intensity fluctuations of the scattered light (Finsy, 1994).

misleading artifacts are frequently present in DLS studies.

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

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

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

in 37º 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.
