Combining Trypsin/Chymotrypsin Enzymatic Digestion

Guidelines for combining trypsin/chymotrypsin enzymatic digestion for proteomics are found below, or can be downloaded as a pdf.

Lipid-storage organelles are stabilized by a number of specific proteins. These proteins are very hydrophobic, which complicates their identification by “classical” proteomic protocols using trypsin digestion. Due to the lack of trypsin cleavage sites, the achievable protein coverage is limited or even insufficient for reliable protein identification. To identify such proteins and to enhance their coverage, we introduced a combination of chymotrypsin and trypsin, which enabled to obtain proteolytic peptides from the hydrophobic regions. This method can be easily applied to identification of other hydrophobic proteins.

Guidelines for sample preparation

  1. Try to avoid any contact of samples and solutions with dust, skin or hair
  2. Clean your bench
  3. Wear gloves at all times
  4. All reagents should be prepared fresh or aliquots could be used if stored at -20°C (the stock solution validity is 6 months if the validity of the reagent itself is not lower)
  5. Use ultra-pure water for all solutions (MilliQ water)
  6. This protocol is optimized for 15μg of protein.

Guidelines for sample submission

  1. Provide 10ul of samples in no recovery vials* or vials with insert for small volumes for LC-MS/MS analysis
    *Autosampler vials appropriate for analysis Waters Total Recovery (part number: 186000385C)
  2. Provide samples in 1.5 ml eppendorf tube for MADI-TOF/TOF analysis.
  3. Label your tube with the sample ID.
  4. Fill in online sample submission form to provide us with more information about your sample.

Solutions of reagents

100% Acetonitrile (CH3CN, HPLC or LC-MS grade)
50% Acetonitrile
-Dilute a volume of 100% ACN 1:1 in MilliQ water
100 mM ammonium bicarbonate (NH4HCO3, MW 79.06)
-0.79 g NH4HCO3 in 100 ml MilliQ water
-Store at -20°C in aliquots of 10ml
50mM acetic acid
-Prepare 50 mM acetic acid solution by adding 287.36 μL of glacial acetic acid to 100 mL of ultrapure water.
50mM ammonium bicarbonate
-Dilute 100 mM NH4HCO3 stock solution 1:1 using MilliQ water
1M DTT (Dithiothreitol, HSCH2(CHOH)2CH2SH, MW 154.24)
- 0.77 g DTT in 5 ml water MilliQ
- Store at -20°C in aliquots of 500 μl
85mM DTT (To reduce the proteins: in-gel reduction is recommended even if the proteins were reduced prior to an electrophoresis run)
-Dilute 1M IAA stock solution using 50mM ammonium bicarbonate
110 mM IAA (Iodoacetamide, C2H4INO, MW184.96)
-Dissolve 68 mg of IAA in 3327 μl of water MilliQ
- Store at -20°C in aliquots of 250 μl
55 mM IAA ( To prevent the re-formation of disulphide bridges)
-Dilute 110mM IAA stock 1:1 using 50mM ammonium bicarbonate

12.5 mg/l of Trypsin from Promega and 12.5mg/l of Chymotrypsin from ThermoScientific
-Trypsin solution: Trypsin 12.5 mg/L in 50 mM ammonium bicarbonate. Dilute the stock solution of trypsin (1 g/L) 80 times with cold (4 °C) 50 mM ammonium bicarbonate to reach the final concentration of trypsin 12.5 mg/L. Prepare the solution immediately before use. Keep it at 4 °C.
Chymotrypsin solution: Chymotrypsin 12.5 mg/L in 50 mM ammonium bicarbonate. Prepare the solution of chymotrypsin as in previous step. Keep the solution at 4 °C.
-Protease mixture solution: Trypsin 6.25 mg/L + chymotrypsin 6.25 mg/L
-Mix equal volumes of trypsin and chymotrypsin solutions in 50 mM ammonium bicarbonate. Keep the solution at 4 °C.

Reduction and alkylation

  1. Typically samples should have a protein concentration of [Protein]=1mg/ml
  2. Take an aliquot of 15μl (15μg of protein)
  3. Reduce with 2μl of 85mM DTT in 50mM ammonium bicarbonate (Ambic)10mM (final concentration) DTT for 40min at 56ºC
  4. Alkylate with 7μl of 55mM IAA in 50mM Ambic 20mM (final concentration) IAA for 30min in the dark at RT
  5. Reduce the sample again with 3μl of 85mM DTT in 50mM Ambic for 10min in the dark at RT, in order to eliminate excess IAA
  6. (Note: only needs to be performed if the proteins are from cell or tissue extraction) Precipitate the sample with 6 volumes of ice-cold acetone or using the 2-D clean-up Kit ( Code number: 80-6484-51, from GE Healthcare) a. Add 162μl of ice-cold acetone, vortex and incubate overnight at -20ºC b. Centrifuge at 15,000 xg for 10 min, in a previous cooled rotor c. Remove the supernatant and allow the pellet to dry for no more than 5min d. Resolubilize the pellet in 27μl of 50mM Ambic.

Trypsin + Chymotrypsin digestion

  1. Add the mixture of trypsin + chymotrypsin in a 1:20 to 1:50 (W/W) ratio regarding the total protein content of your sample. Mix carefully and carry out the digestion for 3 hours at 37 °C.
  2. Stop the trypsin + chymotrypsin digestion by adding up to 5% FA
  3. Dry the digested sample to completion using vacuum centrifuge
  4. Resolubilize the sample peptides:
    4.1 For MALDI-TOF/TOF analysis re-dissolve in 10-20 µl of 0.1% Trifluoroacetic Acid (TFA) and use a Zip Tip to clean up the sample (please see separate protocol)
    4.2 For LC-MS/MS analysis re-dissolve in 10-20 µl of 0.1% of Formic Acid (FA) and use a Zip Tip to clean up the sample (please see separate protocol)