Pharmalyte

Cy2-, Cy3- and Cy5-labeled samples were mixed together, and rehydration buffer was added (7 M urea, 2 M thiourea, 4% CHAPS, 0.5% pharmalytes (GE Healthcare), 40 mM dithiothreitol (DTT), and 0.002% bromophenol blue) to a final volume of 450 μl and used to rehydrate an immobilized pH gradient strip (24 cm; pH 3–10NL; GE Healthcare) by passive diffusion at 20°C for 12h. Isoelectric focusing was performed on an IPG Phor 3 horizontal electrophoresis system (GE Healthcare) with a program of 0.5 kV for 1h, holding at 0.5 kV for 5 h, ramping to 1 kV over 1 h, ramping to 8 kV over 3 h, holding at 8 kV until 60 kV*h, and holding at 0.5 kV for 4 h. Each focused strip was then equilibrated in two steps: 15 minutes in a reducing equilibration buffer (6 M urea, 50 mM Tris-HCl, pH 8.8 with 30% (v/v) glycerol, 2% (w/v) SDS, 0.01 bromophenol, and 10 mg/mL DTT), followed by 15 minutes in an alkylating equilibration buffer where DTT was replaced by 25 mg/mL iodoacetamine. The equilibrated IPG strips were then placed directly on top of polymerized 12% SDS gels and sealed with an agarose sealing solution (25 mM Tris, 192 mM, glycine, 0.1% SDS, 0.5% (w/v) agarose, and 0.02% bromophenol blue). Gels were run in cooled tanks with DIGE buffer on an Ettan Dalt-6 (GE Healthcare) at 1 W per gel until the bromophenol blue dye front reached the bottom of the gel.

GBM U87 cells were treated with 200 μM of TMZ or DMSO for one week or two and subjected to total cellular proteins extraction using lysis buffer containing 4% (w/v) CHAPS (Sigma-Aldrich), 8 M urea (GE Healthcare, Pittsburgh, PA, USA), 1% (v/v) Pharmalytes (GE Healthcare), 2 mg/mL DTT (Sigma-Aldrich), and trace amounts of bromophenol blue (Sigma-Aldrich) followed by sonication on ice. Next, lysates were centrifuged at 12,000 rpm for 1 h at 4 °C. Subsequently, protein concentration of supernatants was determined by the modified Bradford method, and aliquots of protein samples were stored at −80 °C. Differentially expressed proteins were separated using two dimensional gel electrophoresis (2-DE) and mass spectrometry. The 2-DE was performed using 4–7 pI immobiline strips and gels were then subjected to silver staining to visualize proteins. Protien spots of interest were dissected from the gels and the subjected to Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/TOF MS). Experiments were repeated three times.

Samples for 2D proteomic analysis were prepared as follows. Bacterial cultures were grown in LB at 37°C for 18 h and diluted 1:100 with 30 ml of pre-warmed LB. They were further incubated at 37°C with aeration and shaking at 250 rpm until optical density at 600 nm (OD₆₀₀) of 0.85. Cells were then placed on ice for 20 min and harvested at 4°C. Bacterial pellets were resuspended in a sample buffer consisting of 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 1% (w/v) DTT, 2% (v/v) pharmalyte (pH 3.5–10, GE Healthcare, Chicago, IL, United States), and 1 mM benzamidine. These samples were sent to Genomine Inc (Pohang, Korea) for 2D electrophoresis, normalization, and protein identification.

Purified samples were analyzed for charge variants on an iCE3 (Protein Simple) with a FC Column Cartridge (Protein Simple). Samples were prepared by mixing with an ampholyte solution consisting of urea, methyl cellulose, and the appropriate Pharmalyte (GE). Charge variants were detected at 280 nm.

First dimensional protein separation with isoelectric focusing was conducted on a Protean IEF Cell (BioRad, Hercules, CA, United States) with 57 μg of total protein (consisting of 30 μg labeled sample and 27 μg labeled IPS) per immobilized pH gradient (IPG) strip [ReadyStrip IPG Strips 24 cm pH 3-10 non-linear (BioRad)]. The rehydration mix containing both sample and IPS was brought to a final volume of 450 μl and a final concentration of 5 M urea, 0.5%w/v CHAPS, 0.5% v/v Pharmalyte (GE Healthcare), and 12 μl/ml of DeStreak reagent (GE Healthcare). The strips were overlayed with mineral oil (Bio-Rad) and actively rehydrated at 50 V for 12 h and afterward focused by increasing the voltage step by step up to 8000 V within 17 h. The procedure was carried out at 20°C using a current limit of 30 μA per strip. Focused strips were stored at -80°C until further use.