On-bead digestion is done by adding 100 µls of a 0.025 ug/uL solution of MS-grade. Trypsin to the beads. Digest overnight at 37°C. Centrifuge at 5000xg (medium setting, not max) for 3 mins, and retain peptide filtrate.
How do you digest trypsin?
Trypsin Digestion Protocols
- Dissolve the target protein in 6M guanidine HCl (or 8M urea), 50mM Tris-HCl (pH 8), 2–5mM DTT.
- Heat at 37°C for 45–60 minutes.
- For denatured proteins, add 50mM NH4HCO3 or 50mM Tris-HCl (pH 7.8), 1mM CaCl2, until the guanidine HCl or urea concentration is less than 1M.
What proteins does trypsin digest?
Trypsin is a serine protease. It cleaves proteins into peptides with an average size of 700-1500 daltons, which is in the ideal range for MS (Laskay et al., 2013). It is highly specific, cutting at the carboxyl side of arginine and lysine residues.
