In-Gel Trypsin Enzymatic Digestion
Guidelines for in-gel trypsin enzymatic digestion for proteomics are found below, or can be downloaded as a pdf.
Guidelines to protect your samples from contamination with keratin
- 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)
Guidelines for sample submission
- Provide 10ul of samples in recovery vials* or vials with insert for small volumes for LC-MS/MS analysis or 1.5 ml eppendorf tubes for MALDI-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
*Autosampler vials appropriate for analysis - Waters Total Recovery (part number: 186000385C)
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)
Dissolve 0.77 g DTT in 5 ml MilliQ water
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 56 mg of IAA in 3327 µl of MilliQ water
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
20 µg/µl of Trypsin- Pierce Trypsin Protease, MS Grade
(Other enzymes with the same pH tolerance as trypsin can be substituted without modifying conditions. These enzymes includes Chymotrypsin, Asp-N, Glu-C and Lys-C)
Reconstitute lyophilized trypsin using 50mM acetic acid to 1mg/mL (i.e., add 20μL of 50mM acetic acid to 20μg of lyophilized trypsin).
Dilute 1mg/mL trypsin stock solution to 0.01mg/mL using 50mM ammonium bicarbonate. Always work with the trypsin in an ice bucket to prevent auto-proteolysis
Procedure
Excision of protein bands from polyacrylamide gels
- Wash the gel slab with water (2 changes, 10 min each)
- To excise the bands/spots from the gel use clean nitrile gloves and scalpel
- Cut as close to the protein band as possible to reduce the amount of background gel
- Excise a gel piece of roughly the same size from a non-protein containing region of the gel for use as a control
- Cut the gel pieces into roughly 1mm3 cubes
- Put the gel pieces in clean 0.5 or 1.5 ml eppendorfs
De-staining gel pieces from Coomassie stained bands/spot
- Wash the gel pieces with water (15 min shaking)
- Add 100 µl of 100mM 100 mM ammonium bicarbonate (10 min shaking)
- Remove buffer with pipette and add 50 µl of 1:1 50 mM ammonium bicarbonate / ACN to gel pieces (10 min shaking)
- If gel pieces are still blue, repeat steps 2 and 3
- Remove all liquid and add 100% ACN, enough to cover the gel pieces (30 min at 37°C shaking)
- After the gel pieces have shrunk (they become white and stick together) remove the acetonitrile
IMPORTANT: Solvent volumes used in the washing steps should roughly equal 5 times the gel volume
De-staining gel pieces from Silver stained bands/spot
- Add 200ul of 1:1 potassium ferricyanide/sodium thiosulfate and agitate 20 min in the dark at RT
- Remove all the liquid
- Add 200µl of MilliQ water and agitate 20 min at RT
- Remove all the liquid
- Repeat this procedure until the bands/spots are transparent
- Add 30µl of MilliQ water and agitate for 20 min at RT
- Remove all the liquid
- Add 30µl of 100% of ACN and agitate for 30 min at 37°C
- Remove all liquid
IMPORTANT: Solvent volumes used in the washing steps should roughly equal 5 times the gel volume
Reduction and alkylation
- Swell gel pieces in 30 µl of 10mM DTT in 100mM NH4HCO3
- Reduce for 45 min at 56°C with agitation
- Remove excess liquid
- Add 30 µl of 55mM of iodoacetamide in 100mM NH4HCO3
- Alkylate in the dark for 30 min at room temperature with agitation
- Remove all the liquid
- Wash with 100 µl of 100mM NH4HCO3 for 5 min at room temperature and agitation
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Remove all the liquid
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Wash with 100 µl of 100%ACN for 15 min at room temperature and agitation
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Remove all the liquid
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Remove ACN and allow gel pieces to air dry for 5-10 minutes
Trypsin digestion
- Rehydrate the gel pieces with 50μL of 0.01mg/mL trypsin solution to the sample for 30 min at 4°C.
- After 15 min, add more digestion buffer if the initial volume has been absorbed by the gel pieces
- Incubate overnight (16-24 hours) at 37°C
- After overnight incubation, recover the digest to a new tube and add formic acid to each sample to a final formic acid concentration of 5.0% in order to stop the enzymatic reaction.
- Extraction of peptides from the gel (normally used to extract big peptides that don’t leak from the gel easily)
- Add 100 µl of 1% formic acid and incubate for 15 min at RT with shaking, then transfer to the vial containing supernatant
- Add the same volume of 100%ACN and incubate for 15 min at RT with shaking, pipette off and save the supernatant
- Add 100µl of 1:1 ACN/Water, leave for 5 min, then transfer to the vial containing supernatant.
- Add 100µl of 1% formic acid in ACN, leave for 5 min, then transfer to the vial containing supernatant
- Dry the digested sample to completion using the SpeedVac
- Resolubilize the sample peptides:
- For MALDI-TOF/TOF analysis re-dissolve in 10-20 µl of 0.1% Trifluoroacetic Acid (TFA) and use Zip Tip to clean up the sample (please see separate protocol
- For LC-MS/MS analysis re-dissolve in 10-20 µl of 0.1% of formic acid and use Zip Tip to clean up the sample (please see separate protocol)