Ipg pharmalyte ph 3 10

After sample clean‐up, the sample pool was subjected to peptide IEF‐IPG (isoelectric focusing by immobilized pH gradient) in pI range 3–10. The freeze‐dried peptide sample was dissolved in 250 μL rehydration solution containing 8 m urea and allowed to adsorb to the gel strip by swelling overnight. The 24 cm linear gradient IPG (Immobilized PH Gradient) strip (GE Healthcare, Chicago, IL, USA) was incubated overnight in 8 m rehydration solution containing 1% IPG pharmalyte pH 3–10 (GE Healthcare). After focusing, the peptides were passively eluted into 72 contiguous fractions with MilliQ water/35% acetonitrile/35% acetonitrile and 0.1% formic acid, using an in‐house constructed IPG extractor robotics (GE Healthcare Biosciences AB, Uppsala, Sweden; prototype instrument) into a 96‐well plate (V‐bottom, product #651201; Greiner Bio‐One, Kremsmünster, Austria). The BT GSCs samples were rerun and additionally fractionated by IEF‐IPG in pI range 3.7–4.9, to detect more peptides for proteogenomic analyses. The resulting fractions were then dried, frozen, and kept at −20 °C until LC–MS/MS analysis.

The HiRIEF prefractionation method at peptide level was applied as previously described.15 (link) Briefly, after sample clean-up by solid phase extraction (SPE strata-X-C, Phenomenex), the sample pool was subjected to peptide IEF-IPG (isoelectric focusing by immobilized pH gradient) in pI range 3–10 (1 mg). The freeze-dried peptide sample was dissolved in 250 µL rehydration solution containing 8M urea and allowed to adsorb to the gel strip by swelling overnight. The 24 cm linear gradient IPG strip (GE Healthcare) was incubated overnight in 8M rehydration solution containing 1% IPG pharmalyte pH 3–10 (GE Healthcare). After focusing, the peptides were passively eluted into 72 contiguous fractions with MilliQ water/35% acetonitrile/35% acetonitrile and 0.1% formic acid, using an in-house constructed IPG extractor robotics (GE Healthcare Biosciences AB, prototype instrument) into a 96-well plate (V-bottom, Greiner product no 651201). The resulting fractions were then freeze-dried and kept at −20°C until LC-MS/MS analysis and data searches (see online supplementary additional file 1).

After clean-up the sample pools were subjected to peptide IEF-IPG (isoelectric focusing by immobilized pH gradient) in pI range 3–10. Dried peptide samples (300 µg) were dissolved in 160 µL rehydration solution containing 8 M urea, and allowed to adsorb to the gel bridge by swelling overnight. The 24 cm linear gradient IPG strips (GE Healthcare) were incubated overnight in 8 M rehydration solution containing 1% IPG pharmalyte pH 3–10 (GE Healthcare). Samples were applied to the IPG strips by the gel bridge (pH 3.7) at the cathode end and run as described^{44 (link),45 (link)}. After focusing, the peptides were passively eluted into 72 contiguous fractions with MilliQ water using an in-house constructed IPG extractor robotics (GE Healthcare Bio- Sciences AB, prototype instrument) into a 96-well plate (V-bottom, Corning product #3894), which was then dried in a SpeedVac. The resulting fractions were freeze dried and kept at −20 °C.