Ipg strip

After clean up, the TMT‐labeled samples underwent isoelectric focusing (IEF) on four 24‐cm, 3.7–4.9 immobilized pH gradient (IPG) strips (GE Healthcare, Uppsala, Sweden). Briefly, samples were rehydrated in 8 m urea with bromophenol blue and 1% Pharmalyte (GE Healthcare), loaded onto IPG strips, and separated, according to previously published protocols [35]. The IPG strips were subsequently subjected to passive elution with MilliQ water into 72 fractions by using an in‐house robot. The obtained fractions were dried with a SpeedVac and stored at −20 °C.

Proteins were extracted from cells using a urea lysis buffer (7 M urea, 2 M thiourea, 65 mM CHAPS, 0.5 M EDTA, 50 mM Tris, 0.01% BPB, and 65 mM DTT) supplemented with protease inhibitors (Roche), 200 mM PMSF (phenylmethylsulfonyl fluoride), and ampholytes. Cell lysates were desalted and concentrated using Amicon ultra centrifugal filters (Merck Millipore, Darmstadt, Germany), and the resulting protein concentration was measured using a Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions.
Proteins were resolved by 2DE, which separates proteins based on isoelectric point (first dimension) and size (second dimension). For isoelectric focusing (IEF), each protein sample was loaded on an IPG strip (pH 3–10 NL; 130 mm × 3 mm × 0.5 mm, GE Healthcare), after which the strip was rehydrated for 18 h. After performing the IEF electrophoresis step for a total of 45,000 Vhrs, the IPG strip was first soaked in equilibration buffer consisting of 0.5 M Tris pH 8.8, 6 M urea, 2% SDS, and 30% glycerol containing 100 mM DTT for 15 min, and then in equilibration buffer containing 110 mM iodoacetamide (IAA) for 15 min. For the second dimension, proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Colloidal Coomassie blue staining was used to visualize the separated protein spots.

Prior to isoelectric focusing, IPG strips (pH 4–7, 13 cm; GE Healthcare, Little Chalfont, UK) were passively rehydrated with 250 µl of protein solution in wells for 14 h. Isoelectric focusing was conducted using the following protocol: 250 V for 15 min, 500 V for 2 h, gradient voltage increased to 1 000 V for 1 h, gradient voltage increased to 8 000 V for 2,5 h, 8 000 V for 3 h, and finally reduced to 500 V (Ettan IPGphor3; GE Healthcare, Little Chalfont, UK). To prepare for the second-dimension SDS-PAGE, strips were incubated in equilibration buffer (50 mM Tris–HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS and 0.002% Bromophenol Blue) for two 15 min periods, first with 1 g l⁻¹ dithiothreitol and then with 48 g l⁻¹ iodoacetamide. IPG strips were placed on top of 12% polyacrylamide gels, which were run in thermo-regulated electrophorese unit at 10 °C (SE 600 Ruby; Amersham Biosciences, Amersham, UK) at 10 mA per gel for 1 h and then 30 mA per gel until complete migration. Gels were subsequently stained with “Blue Silver” (Candiano et al., 2004 (link)) and destained with Milli-Q water for 48 h. The resulting gels were scanned with a transparency scanner (Epson Perfection V700; Epson, Suwa, Nagano, Japan ) in gray scale with 16-bit depth and a resolution of 400 dpi.