In-Gel Digestion of Proteins for MS
Before cutting the gel pieces wash the gel well
with water to remove acetic acid, methanol, and SDS and buffer salts.
Cut gel spots and take them in 500microL eppendorf tube.
Add some (100-200 microL) 50%AcCN/50% 50mM Ammonium Bicarbonate,
pH8 to it and crush the gel pieces using a clean pestle (see notes below) and leave for
15 minutes.
Centrifuge and remove supernatant and add another aliquot of the
above 50% AcCN/Ammn. Bicarb solution vortex and leave 15 min at room T.
Centrifuge repeat above step.
Hopefully all the dye is gone by now.
To the left over gel pieces add 100 microL AcCN and vortex and
speed vac.
Reduction and Alkylation of cysteine residues
(carbmidomethylation)
If you have done this before running the gel than proceed to
digestion directly.
To the gel pieces add enough 5mM DTT solution (in ammonium
bicarbonate, 50mM pH8) to soak the gel pieces and incubate at 37C for
30mins. Wash with 50% acetonitrile in
ammonium bicarbonate with 100microL twice.
Add 100microL acetonitrile and vortex and speedvac to dryness. Now add 10mM Iodoacetamide solution to the
gel pieces (enough to soak the gel pieces) and incubate for 30mins at room
temperature. Wash twice as above and
dry the gel pieces in acetonitrile and proceed for digestion step.
Take Promega Trypsin in 50mM Ammonium Bicarbonate pH8 (stock
solution - 20micrograms in 1mL buffer) and add to the gel slices so that the
gel slices are just covered with a thin layer of the solution. Note don't add too much solution (you will
see to many trypsin peaks.)
Leave at 37C overnight.
Add 50% AcCN/0.3%TFA in water to extract the peptides (Volume
roughly the same amount of gel you have in there.)
Leave for 15min and spin and take supernatant and save.
Add another 100microL of the above AcCN solution and leave for
15min spin and extract supernatant and save (In the same tube as above)
If you want you can extract for the third time.
The mixed supernatants can now be speedvac and run on MALDI. I used to speedvac and add water and
speedvac again and repeat the process three times to chase the ammn. bicarb.
away.
Before using the pestles clean them well with ddH2O otherwise you
see a lot of keratin peaks due to the dust on the pestles. Please note wear gloves when handling the
gel and washing the pestles and through out the digestion and extraction
procedure.
Ordering information:
Pestle
Vendor Fisher
Scientific
Phone # 800 766
7000
Description Kontes
Pellet Pestle, 0.5mL
Catalog# K749521-0590
DTT
Sigma
D-0632
Iodoacetamide
Sigma I-1149
There are several other ways of reducing the cysteines and
alkylating them. For reduction another
wellknown reagent is triphenylphosohine and for alkylation, vinyl pyridine
(this mode of alkylation helps in the MALDI, in the sense that the modification
helps cysteine containing peptides to fly well.) The vinyl pyridine reagent induces pyridyl ethylation
modification of the cysteines. Care has
to be taken to keep the vinyl pyridine reagent dry. The DTT solution can be made up in stock and frozen over a period
of time. But the alkylating agents have
to be made fresh every time the digestions are being done (both iodoacetamide
and vinyl pyridine.)