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IN SOLUTION DIGESTION
LCMS+ fractions, reduction alkylation, trypsin digestion:
To 10 to 20 µL aliquots:
Add 15 µL 10mM DTT in 50mM ammonium bicarbonate. 60 min, 370C.
Add 15 µL 50mM iodoacetamide in 50mM ammonium bicarbonate. 60 min, 370C.
Add 0.1µg trypsin in 50mM ammonium bicarbonate. 3 hours, 370C.
Please note:
1. Trypsin aliquots are kept dried down. (Do not store any trypsin solutions.)
2. For a known amount of protein the ratio should be 1:40 trypsin:protein.
3. Iodoacetamide amount (in moles) should be > 2 times DTT.
TAP fractions, no gel, reduction alkylation, trypsin digestion:
For soluble fraction follow the above protocol, spin after the DTT step and keep pellet.
Pellet:
Dissolve in 8 µL 6M guanidine HCl in 200 mM ammonium bicarbonate. µL
Add 1 µL DTT (final 10mM)
Add 1 µL iodoacetamide (final 50 mM)
Add 50 µL trypsin, 0.1 µg.
Keratin Contamination:
Keratin
contamination could be a major problem and appear as the main protein
candidate in LCMSMS data. The source of contamination usually lies
prior to digestion or is introduced during digestion.
Avoiding Keratin Contamination:
- Wear Gloves - Keratin is abundant in hair, nails, skin etc.
- use a Laminar flow hood if you can.
- Do not keep the tubes exposed to air for long periods of time during digestion.
- Do not handle the gel with you bare hands.
- Make fresh solvents.
- See a digestion protocol in a video (from UC Davis)
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