Guidelines for in-solution chymotrypsin enzymatic digestion for proteomics are found below, or can be downloaded as a pdf.
Guidelines for sample preparation
- Try to avoid any contact of samples and solutions with dust, skin or hair
- Clean your bench
- Wear gloves at all times
- 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)
- Use ultra-pure water for all solutions (MilliQ water)
- This protocol is optimized for 15μg of protein.
Guidelines for sample submission
- 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)
- Provide samples in 1.5 ml eppendorf tube for MADI-TOF/TOF analysis.
- Label your tube with the sample ID.
- 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)
-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
25 µg/µl of Chymotrypsin Endoproteinase MS Grade, ThermoScientific (Other enzymes with the same pH tolerance as trypsin can be substituted without modifying conditions. These enzymes include Trypsin, Asp-N, Glu-C and Lys-C)
-Reconstitute lyophilized chymotrypsin using 50mM acetic acid to 1mg/mL (i.e., add 25μL of 50mM acetic acid to 25μg of lyophilized chymotrypsin).
-Dilute 1mg/mL chymotrypsin stock solution to 0.01mg/mL using 50mM ammonium bicarbonate.
-Always work with the chymotrypsin in an ice bucket to prevent auto-proteolysis
Reduction and alkylation
- Typically samples should have a protein concentration of [Protein]=1mg/ml
- Take an aliquot of 15μl (15μg of protein)
- Reduce with 2μl of 85mM DTT in 50mM ammonium bicarbonate (Ambic)10mM (final concentration) DTT for 40min at 56ºC
- Alkylate with 7μl of 55mM IAA in 50mM Ambic 20mM (final concentration) IAA for 30min in the dark at RT
- 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
- (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.
- Add 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 overnight at 37 °C.
- Stop the chymotrypsin digestion by adding up to 5% FA
- Dry the digested sample to completion using vacuum centrifuge
- 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)