Destaining, reduction and alkylation

Solutions

Destaining solutions
(prepare about 50 mL of each solution in advance)

25 mM NH4HCO3

Wash1
25% ACN in 25 mM NH4HCO3

Wash2
50% ACN in 25 mM NH4HCO3

Reduction solution
50 mM DTT in 25 mM NH4HCO3 prepared by diluting 1 M DTT in water (prepared in advance and stored in -20 °C)

Alkylation solution
55 mM (about 10 mg/mL) iodoacetamide in 25 mM NH4HCO3, prepared fresh

Trypsin batch solution
(kept in -20 °C in 10–20 µL aliquots)
25 µg trypsin in 250 µL 10 mM HCl

Trypsin application solution
Trypsin batch solution and 25 mM NH4HCO3 mixed 1:9

Procedure

  • Clean glass plate with water, technical ethanol and miliq water, dry.
  • Place the gel on plate, cut the bands of interest with a clean scalpel, cut every band in parts of about 2 mm in size, place these parts in fresh eppendorf tube.
  • Add 200 µL of Wash1, shake in 37 °C at 1100 rpm for 15 min, remove liquid
  • Add 200 µL of Wash2, shake in 37 °C at 1100 rpm for 15 min, remove liquid
  • Add 200 µL of Wash1, shake in 37 °C at 1100 rpm for 15 min, remove liquid
  • Add 200 µL of Wash2, shake in 37 °C at 1100 rpm for 15 min, remove liquid
  • Add 200 uL of reduction solution, keep at 37 °C for 45 min, remove liquid
  • Add 200 uL of alkyklation solution, keep at RT for 2 h in dark, remove liquid
  • Add 200 µL Wash2, shake at RT for 15 min at 1100 rpm, remove liquid
  • Add 200 µL Wash2, shake at RT for 15 min at 1100 rpm, remove liquid
  • Add 50 µL of acetonitrile, keep 15 min at RT, remove liquid
  • Dry 10 min in speedvac, at RT
  • Add 15 µL trypsin application solution, wait 30 min
  • Add 25 µL NH4HCO3 (as much as needed to cover gel pieces)
  • Keep overnight at 37 °C

Notes

If proteins were previously alkylated, simply omit reduction and alkylation steps.
This procedure works for silver staining, coomassie staining and SYPRO Ruby staining as well.

Extraction and MS analysis
(using Dionex Ultimate 3000 NanoRSLC HPLC connected to Bruker MicroTOf-Q II MS with NanoESI source with New Objective 10 µm id emitters)

After digestion

  • Sonicate 30 min on ultrasonic bath
  • Add 3.5 µL 10% TFA
  • Sonicate 30 min on ultrasonic bath
  • Transfer liquid to a low-binding eppendorf tube (e.g. Sigma Clearviev)
  • Add 50 µL acetonitrile to gel pieces, leave for about 15 min, until they shrink
  • Combine acetonitrile and water extracts, evaporate to dryness in speedvac
  • Resuspend in 17 µL 2% acetonitrile, 0.05% TFA (loading buffer for trap-column), vortexing for 15 minutes and brief sonication
  • Load 11 µL on a trap column (e.g. Acclaim PepMap100 C18 Nano-Trap (Dionex)) with 5 µL/min for 6 min
  • Resolve peptides with 30 min linear gradient 2–40% acetonitrile in 0.05% formic acid, followed by wash and blank run to avoid carryover

Contributed by Artur