**2.13 Western blot**

334 Gel Electrophoresis – Advanced Techniques

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.

HDL (500 µg) was lyophilized and dissolved in 100 µL 2-DE sample buffer (described earlier).The sample was then incubated with 20 U of PNGase F (Sigma-Aldrich, P7367) in 37

HDL was desalted in a PD-10 column and eluted in a 50 mM Na2HPO4, 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 (NH4HCO3, 12mM, pH 7.1) and

After digestion and drying of peptide samples, some were desalted and purified by the use of ZipTipC18® 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

Peptides obtained after digestion were mixed 1:1 with matrix, α-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 ZipTipC18® 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).

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

washed with 5x10 µL of 0.1% TFA before peptides were eluted with 10 µL 50% ACN.

C over night. After incubation, sample were stored in -20ºC until 2-DE.

subsequently frozen in -70ºC. Sample was lyophilized before 2-DE.

**2.9.5 Enzymatic deglycosylation** 

**2.10 ZipTip** 

**2.11 Mass spectrometry** 

**2.12 Database search** 

Fragmentation spectra were interpreted manually.

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).
