Ipg pharmalyte

The pre-fractionation was done using HiRIEF (32 (link)). The pooled sample (450 μg) was dissolved in 250 μl of 8 m urea and 1% IPG pharmalyte (broad range pH 3–10, GE Healthcare, Chicago, Illinois), and the IPG drystrip was rehydrated overnight. The peptides were focused on the gel strip based on their isoelectric point and eluted into 72 contiguous fractions, as described previously (24 (link)).

The prefractionation method was applied as previously described in ref. ^{103 (link)}. Sample pools were subjected to peptide IEF-IPG (isoelectric focusing by immobilized pH gradient) in the pI range 3–10 and 3.7–4.9. Dried peptide samples were dissolved in 250 µL rehydration solution containing 8 M urea, and allowed to adsorb to the gel bridge strip by swelling overnight. The 24 cm linear-gradient IPG strips (GE Healthcare) were incubated overnight in an 8 M rehydration solution containing 1% IPG pharmalyte pH 3–10 or 2.5–5, respectively (GE Healthcare). After focusing, the peptides were passively eluted into 72 contiguous fractions with MilliQ water/ 35% acetonitrile (CAN)/ 35% ACN + 0.1% formic acid (FA) using an in-house constructed IPG extractor robotics (GE Healthcare Bio-Sciences AB, prototype instrument) into a 96-well plate (V-bottom, Greiner product #651201), which were then dried in a SpeedVac. The resulting fractions were dried and kept at −20 °C.