In-gel Trypsin digestion protocol and MALDI spotting

Solutions needed:

100% Acetonitrile

100 mM Ammonium Bicarbonate

Wash Solution: 50% acetonitrile and 50 mM Ammonium Bicarbonate

Reduction solution: 10 mM DTT in 100 mM Ammonium Bicarbonate

Alkylation solution: 50 mM Iodoacetamide in 100mM Ammonium Bicarbonate

Trypsin solution: 20 μg/ml

Extraction solution: 0.1% TFA and 50% Acetonitrile

 

Excise stained gel piece(s), mince into 1 mm3 pieces and transfer into a sterile microcentrifuge tube.

Wash gel with 500 µL of wash solution (50% acetonitrile, 50 mM ammonium bicarbonate) and incubate   at room temperature for 15 min with gentle agitation (vortex mixer on lowest setting). Remove solution with a pipette.

Wash gel two more times with 500 µL of wash solution (15 min each) or until the Coomassie dye has been completely removed.

Dehydrate the gel in 100% acetonitrile for 5 min. When dehydrated, the gel pieces will have an opaque white color and will be significantly smaller in size.

Remove acetonitrile with a pipette and then completely dry gel at room temperature for 10-20 min in a centrifugal evaporator.

Rehydrate gel pieces in 150 µL reduction solution (10 mM DTT, 100 mM ammonium bicarbonate) for 30 min at 56 C.

Discard reduction solution with a pipette and add 100 µL alkylation solution (50 mM iodoacetamide, 100 mM ammonium bicarbonate) and incubate for 30 min in the dark at room temperature.

Discard alkylation solution with a pipette and add 500 µL of wash solution and incubate at room temperature for 15 min with gentle agitation.

Discard wash solution and dehydrate gel in 100 µL 100% acetonitrile for 5 min.

Discard acetonitrile and completely dry gel at room temperature in a centrifugal evaporator.

While gel is drying prepare protease digestion solution. Typically, this is modified sequencing grade trypsin (Product number V5111, Promega, Madison, WI). Resuspend lyophilized trypsin (20 µg/vial) in 1 mL of 50 mM ammonium bicarbonate, aliquot (50 µL/tube) and store at -70 C. Do not freeze-thaw more than once.

Rehydrate the gel with a minimal volume of protease digestion solution. Use 20 µL for small gel plugs. Add more if gel pieces absorb all the liquid. Gel pieces must be hydrated throughout the digest. Digest overnight at 37 C.

---------------------------------Overnight---------------------------------------------

Spin down sample by centrifugation (12 k g for 30 sec).

Transfer supernatant (containing tryptic peptides) to sterile centrifuge tube.

Add 25-50 µL of extraction solution (60% acetonitrile, 0.1% TFA) to gel pieces and sonicate in ultrasonic waterbath for 10 min. Alternatively, agitate gently by vortexing at lowest setting.

Spin down sample by brief centrifugation (12 k g for 30 sec).

Transfer supernatant (containing additional tryptic peptides) to tube from step 14.

Extract the gels with an additional 25-50 µL of extraction solution. Agitate gel pieces by sonicating in a waterbath for 10 min or with gentle vortexing.

Spin down sample and transfer supernatant to tube from step 14.

Dry the pooled extracted peptides by centrifugal evaporation to near dryness. Do not use heat. Do not dry for extended time.

Add 5 µL of resuspension solution (50% acetonitrile, 0.1% TFA) to each tube and sonicate tube in water bath or gently agitate on a vortex at lowest setting.

Spin down sample and spot 0.5 µL on MALDI plate followed by 0.5 µL of alpha-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 50% acetonitrile, 0.1% TFA).

Allow spots to dry completely. Load plate into Voyager.

Calibrate using internal tryptic peaks of 842.5 and 2211.1 Da.

Notes

This protocol contains a reduction and alkylation step. Alternatively, this can be performed prior to gel electrophoresis or after first dimension isoelectric focusing.

After peptide extraction mass spec analysis should be performed as soon as possible.

Preparation of peptides must be performed with labware that has never been in contact with nonfat milk, BSA, or any other protein blocking agent to prevent carryover contamination.

Always use non-latex gloves when handling samples, keratin and latex proteins are potential sources of contamination.

Never re-use any solutions, abundant proteins will partially leach out and contaminate subsequent samples.