Trypsin digestion

Reagents:
Buffer A: 400 mM ammonium bicarbonate/8 M Urea
Reduction: 45 mM DTT in water
Alkylation: 100 mM iodoacetamide in water
Trypsin solution: 1 µg/µl Trypsin (sequencing grade and modified, Promega) in resuspension buffer

Tools:
Vortexer
(Siliconized) 0.5 ml microfuge tubes
Incubator or water bath capable of maintaining 37ºC and 52ºC
Speed-Vac Concentrator

Procedure:
1. Prepare a protein-free negative control by starting with ultra-pure water of the same volume as the samples
2. Prepare a positive control using the same volume as the samples; the positive control should be prepared at approximately 1 to 2 pmol/µl.

Reduction and Alkylation:
1. Sample should start out in an aqueous solution without detergents or organics.
2. Place samples in a speedvac at 60 ºC until samples are almost dry (<
5 µl)
3. Add 50 µl 400 mM ammonium bicarbonate/8 M Urea solution
4. Add 10 µl 45 mM DTT and incubate 15 min at 52 ºC
5. Allow samples to cool to room temperature before proceeding
6. Add 10 µl 100 mM iodoacetamide incubate 10 min at RT

Digestion:
1. Prepare Trypsin solution and allow it to incubate for 20 min at room temperature to allow time for activation.
2. Add 1 µl trypsin solution to each sample.
3. Incubate overnight at 37ºC (16-24h usually).

References
Matsudaira, Paul (ed.) A Practicle Guide to Protein and Peptide Purification for Microsequencing, 1993, Academic Press, New York, p. 55-56