Sequencing grade endoproteinase lys c

Gel plugs were removed from the gel using a Pasteur glass pipette, placed into low binding tubes and then destained using 50 μL of 50 mM ammonium bicarbonate/50% (v/v) ACN for 30 min at 37 °C. The plugs were then incubated with 10 mM dithiothreitol (DTT) for 60 min at 60 °C. The DTT was then discarded and 55 mM iodoacetamide (IAM) stock solution was added to each tube and incubated for 45 min at room temperature in the dark. After discarding the IAM, the plugs were washed twice using 50 mM ammonium bicarbonate/50% (v/v) ACN. The plugs were then dehydrated by adding 10 μL of 100% ACN. Sequencing grade endoproteinase Lys C (Wako) (diluted in 25 mM Tris-HCl, 1 mM EDTA, pH 8.5) was then added and the digests incubated overnight at 37 °C. The reaction was stopped by adding formic acid solution to a 1% final concentration (v/v).

Protein samples were phosphorylated in vitro and incubated with PreScission protease (GE Healthcare) overnight at 4°C. The phosphorylated protein was separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). The band was excised, washed, destained, reduced, and alkylated with iodoacetamide and digested with sequencing-grade endoproteinase Lys-C (Wako Chemicals). The digest was desalted on a C18 Zip-Tip (Merck Millipore), mixed with matrix (ACHA), spotted onto a plate (dried-droplet method) and analyzed on a 4700 TofTof (Thermo Fisher Scientific).