Trypsin in-gel digestion of proteins

(adapted from Shevchenko et al.,1996)

Always wear gloves to avoid keratin contamination.

This protocol is compatible with mass spectrometric protein analysis. Generally two days are required for completion of the analysis. On the first day samples are prepared for overnight digestion. The following day samples are lyophilised and then reconstituted in a solution for MS analysis.

We have two methods for protein analysis. One method is short and is practically always used for protein identification purposes. The second method is longer, and involves reduction and alkylation of cysteine-containing peptides. This is useful when a higher coverage of the protein is required, e.g. for structural analysis where more of the protein is identified. Both the short and the long methods are described below.



Trypsin digestion - short method (ID purposes only)

 

  1. Excise protein spot/band and dehydrate in CH3CN for approximately 10 minutes. Remove CH3CN and SpeedVac until dry.
  2. Re-swell gel pieces at 4oC for 45 minutes in buffer containing trypsin and 50 mM NH4-HCO3. (Approx. 5 µL/mm2 gel). The gel pieces should just be covered. Suggested amount of trypsin is 12.5 ng/µL of buffer for proteins that have been silver stained. Digest overnight at 37 oC (or at least for 3 h). *Note: NEVER use more than 1 ug of trypsin per sample for Mass Spec analysis.
  3. Centrifuge gel pieces and collect supernatant. Further extract peptides by one change of 20mM NH4-HCO3, centrifuge then collect, and 3 changes of 5% formic acid in 50% CH3CN (20 minutes between changes) at room temp.
  4. Dry sample down in speedvac until desired volume has been reached. 

 

Trypsin digestion - long method (higher coverage, includes reduction and alkylation)

  1. Excise protein spot/band and dehydrate in CH3CN for approx. 10 min. Remove CH3CN and SpeedVac until dry. Cover gel pieces with 10 mM DTT in 100 mM NH4-HCO3.  Reduce proteins for 1 h at 56 C.
  2. Cool to room temp, remove DTT solution and add equal volume of 55 mM iodoacetamide in 100 mM NH4-HCO3. Incubate for 45 minutes in dark place at room temp.
  3. Wash gel pieces with 50-100 ul aliquots of 100 mM NH4-HCO3 for 10 min. Dehydrate with CH3CN, re-swell in 100 mM NH4-HCO3, and shrink again with CH3CN.
  4. Remove liquid phase and speedvac.
  5. Re-swell gel pieces at 4 oC for 45 min in buffer containing trypsin and 50 mM NH4-HCO3. (Approx. 5 µL/mm2 gel). The gel pieces should just be covered. Suggested amount of trypsin is 12.5 ng/µL of buffer for proteins that have been silver stained. Digest overnight at 37 oC (or at least for 3 h). *Note: NEVER use more than 1 ug of trypsin per sample for Mass Spec analysis.

  6. Centrifuge gel pieces and collect supernatant. Further extract peptides by one change of 20mM NH4-HCO3, (centrifuge then collect), and 3 changes of 5% formic acid in 50% CH3CN (20 min between changes) at room temp.
  7. Dry sample down in speedvac until desired volume has been reached.

 

Calculations

50 mM NH4-HCO3: 79.06 mg/mmol x (50mM/1000ml) = 3.95 mg/ml

make 10x (500mM NH4-HCO3) = 39.5 mg/ml