This protocol assumes that the samples are in an appropriate buffer and total protein quantification performed to ensure that the starting material for each sample is equal.
- Dissolution buffer: 0.5 M triethylammonium bicarbonate (TEAB)
- Reducing Reagent: 50 mM tris-(2-carboxyethyl) phosphine
- Cysteine Blocking Reagent: 200 mM methyl methanethiosulfonate in isopronanol
1. To each of up to eight samples tubes (each containing 20 to 100 µg protein), top up each sample to 20 µl using the appropriate sample buffer.
2. To each sample tube, add 2 µl of Reducing Reagent.
3. Vortex to mix, then briefly centrifuge.
4. Incubate the tubes at 60oC for 1 hour.
Important tip: If your sample buffer contains urea, incubate the tubes at 37oC for 1 hour instead.
5. Briefly centrifuge to bring the sample to the bottom of the tube
6. To each tube, add 1 µl of Cysteine Blocking Reagent. This step can be performed in the fume hood as the odour of the reagent might be unpleasant.
7. Vortex to mix, then briefly centrifuge.
8. Incubate the tubes at room temperature for 10 minutes.
Important: If sample buffer contains chemicals which may interfere with trypsin activity (e.g. chaotropic agents, detergents or surfactants), dilute using 50 mM TEAB to reduce the chemicals to trypsin compatible levels. For instance, trypsin is compatible with 1 M urea and 0.05% SDS.
9. Reconstitute sufficient vials of trypsin with 25 µl of ultrapure water each.
Recommendation: 1 vial of trypsin for two samples (of 100 µg each)
10. Vortex to mix, then briefly centrifuge.
11. To each sample, add 10 µl of the trypsin solution.
12. Vortex to mix, then briefly centrifuge.
13. Incubate the tubes at 37oC overnight (12 to 16 hours).
14. Briefly centrifuge to bring the protein digest to the bottom of the tube.
15. Evaporate the rest of trypsin-digested samples to dryness.
QC Checkpoint: Aliquots from before and after digestion can be analysed using SDS-PAGE to verify complete digestion. If any sample shows incomplete digestion, trypsin digestion should be repeated.