Ipgphor system

Two‐dimensional (2D) electrophoresis (IEF/SDS‐PAGE‐R) of the cell membranes of HepG2 was carried out as follows, according to Oliveira et al. (2019 ). It was applied in the first dimension, isoelectric focusing (IEF), 1,400 µg of HepG2 membrane protein, separated using the IPGphor system (Amersham Pharmacia).
The second dimension (SDS‐PAGE‐R) was performed after IEF. The gel strips were thawed and equilibrated for 15 min, with stirring, in 50 mM Tris‐HCl buffer, pH 8.8, containing 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, and 1% (w/v) dithiothreitol. The strips were subsequently equilibrated for another 15 min, with agitation, in a similar solution that contained 2.5% (w/v) iodoacetamide instead of dithiothreitol, placed on top of a 17.5% (w/v) acrylamide SDS‐PAGE gel, sealed with 0.7% (w/v) agarose (containing 0.002% (w/v) bromophenol blue), and electrophoresed (220 V, 15 mA for 15 min followed by 220 V, 30 mA). After electrophoresis, the gels were stained by silver nitrate (Blum et al., 1987 ).

HMVEC-L cells were harvested by centrifugation and then disrupted with lysis buffer containing 5 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% Triton X-100, and 2 mM PMSF. The cell lysate was centrifuged at 12,000 Χ g for 30 min, and the supernatant fraction was collected. Protein concentrations were determined using a BCA assay kit (Pierce). Immobiline DryStrips (Amersham Biosciences) were used for isoelectric focusing, which was carried out with 1 mg of the extracted protein on an IPGphor system (Amersham Biosciences). After IEF separation, the proteins were separated in the second dimension by SDS-PAGE. For image analysis, the gels were visualized with Coomassie Brilliant Blue G-250 according to the manufacturer’s instructions. The 2-D gels were scanned with an ImageScanner (Amersham Biosciences) in transmission mode. Spot detection and matching were performed using ImageMaster 2D version 5.0 (Amersham Biosciences). Digitized images were analyzed using the ImageMaster program to calculate the 2-D spot intensity by integrating the optical density over the spot area (the spot “volume”) and normalized. The values were normalized and then exported to SPSS 18.0 for statistical analysis.

The pooled iTRAQ labeled samples were dissolved in 300uL 1% Pharmalyte (Amersham Biosciences) and 8M urea and evenly coated on pre-hydrated IPG strips (pH 3-8). The peptides were isolectrically focused successively for 1h at 500v, 1h at 1000v, 1h at 3000v, and 8.5h at 8000v to give a total of 68kv in an IPGphor system (Amersham Biosciences). The peptides were extracted using an acetonitrile (ACN) and formic acid solution. Fractions were lyophilized in a vacuum concentrator and purified on an SPE C18 column. The purified fractions were re-lyophilized and stored in -20°C for use in mass spectrometric analysis.

The volume of the proteins extracts was adjusted to a final protein concentration of 500 μg in 340 μl of rehydration buffer. Subsequently, 1% DTT and 2% immobilized pH gradient (IPG) buffer pH 3–10 (Amersham Biosciences, Freiburg, Germany) were added. The protein sample was loaded onto 17- cm, non- linear pH 3–10 IEF isoelectric focusing strips (Immobiline DryStrip, Amersham Biosciences). IEF was performed using a IPGphor™ system (Amersham Biosciences) with the following program: 10 h at 20°C, 12 h at 30 V, 1 h at 500 V, 8 h at 1,000 V and 10 h at 8,000 V.
The strips were equilibrated for 15 min in 10 ml of equilibration solution (0.375 M Tris–HCl, pH 8.8, 6 M urea, 20% [v/v] glycerol and 2% [w/v] SDS), with 2% (w/v) DTT and for 15 min in 10 ml of equilibration solution containing 2% (w/v) iodoacetamide. Subsequently, the strip was transferred onto a 10% SDS-PAGE gel for second-dimension electrophoresis using a Bio-Rad Protean II system with 30 W per gel. The gels were subsequently stained with Coomassie brilliant blue.

For O-glycosylation detection, 100 micrograms of pooled concentrated urines of 8 ApoA-Ib positive FSGS recurrent patients was resolved in a 7-cm immobilized pH gradient strip (Immobiline DryStrip 3–10; Amersham Biosciences Europe, Freiburg, Germany) and focused in an IPGphor system (Amersham Biosciences Europe). Once focused, the strips were sequentially reduced and alkylated in SDS buffer, placed on a SDS-PAGE gel and run. Afterwards the gel was stained with proQ emerald 300 (Thermofisher scientific) following the manufacturer instructions. For N-Glycosilation study, 40 micrograms of concentrated urine of three ApoA-Ib positive FSGS recurrent patients were treated with PNGase F (New England Biolabs Inc. Ipswich, MA, USA) following the manufacturer protocol. After PNGase F treatment the samples were run in 15% SDS-PAGE gels and transferred to PVDF membranes and proved with anti-ApoA-I (PAB8546, Abnova, Taipei, Taiwan) and anti-transferrin (BioVision Incorporated, Milpitas, CA, USA) as a control of the enzyme activity. Chemiluminiscence was detected using 2 minutes exposure on the Odyssey FC detector (LI-COR Biosciences) and the obtained images were processed with the Image Studio Lite software (LICOR Biosciences). Brightness and contrast parameters were adjusted across the entire image only when needed.