MS sample preparation

MS sample prep

• Wash the beads 2x with 1ml 50mM Tris pH 7.5 (transfer to new tube with first wash)
• Wash the beads 2x with 1ml Urea wash buffer 2 (50mM Tris pH 7.5, 2M Urea)
• Remove the buffer and add 80µl Trypsin buffer (50mM Tris pH 7.5, 1M Urea, 1mM DTT, 0.4µg Trypsin)
• Incubate for 3h at 25°C with shaking
• Transfer the supernatant to a fresh tube
• Wash the beads 2x with 60µl ½ Urea wash buffer 2 (50mM Tris pH 7.5, 1M Urea) and combine the supernatants with the Trypsin digest supernatant
• Reduce the eluate by adding DTT to a final concentration of 4mM (1.6µl 500mM DTT to 200µl eluate) and incubating at 25°C for 30min with shaking
• Alkylate the eluate by adding Iodoacetamide to a final concentration of 10mM (4µl 500mM IAM to 200µl eluate) and incubating at 25°C for 45min with shaking
• Add 0.5µg Trypsin (0.5µl of 1µg/µl stock) and complete the digestion by incubating at 25°C over night (14.5h) with shaking
• Add formic acid to a final concentration of ~ 1% (2.05µl 100% formic acid)
• Desalt samples on C18 tips (all pipetting steps very slow and without getting air into the column material!)
  • Prepare a 0.65ml tube with 200µl buffer B2 (0.1% formic acid, 50% acetonitrile) for the elution
  • Activate a 100µl C18 desalting tip (2x): Aspirate 200µl buffer B2 and discard solvent
  • Equilibration (4x): Aspirate 200µl buffer A2 (0.1% formic acid) and discard solvent
  • Peptide binding (8x): Aspirate and dispense the peptide sample
  • Washing (10x): Aspirate 200µl buffer A2 and discard solvent
  • Elution: Aspirate 200µl buffer B2 from the prepared tube and pipette up and down (8x)
  • Dry the peptides in the Speed Vac