In-solution Digestion Protocols
In-Solution Reduction/Alkylation Digestion
- Lyophilize protein sample (10-20µg).
- Dissolve protein pellet in 40µL 8M Urea (fresh and dissolved in 100mM Tris-HCl, pH 8.5 (denatures proteins).
- Add 0.25µL of 500mM TCEP (Tris(2-CarboxyEthyl) Phosphine) in LC-MS water (reduces disulfides).
- Incubate at room temperature for 20 minutes.
- Add 0.9µL of 500mM iodoactamide (fresh and dissolved in LC-MS water) and incubate for 15 minutes at room temperature in the dark (alkylates cysteines to prevent disulfide bond formation and their effect on peptide sequencing).
- Add 1µL of 0.1µg/µL of Lysyl endopeptidase in 100mM Tris-HCl pH 8.5 (from Achromobacter lyticus with cleavage occurring at lysine and is more stable than Trypsin under high urea denaturing conditions).
- Incubate for 4 hrs. at room temperature in the dark.
- Dilute sample to final concentration of 2M urea by adding 120µL of 100mM Tris-HCl pH 8.5 (dilutes denaturant to a suitable concentration for trypsin activity).
- Adjust to 1mM CaCl2 by adding 1.6µL of 100mM CaCl2 (enhances trypsin activity).
- Add 1µL of 0.5µg/µL trypsin in water (trypsin cuts the protein backbone after Arg/Lys from C- to N- terminus).
- Incubate in the dark overnight at room temperature.
- Adjust/dilute solution to a 5% formic acid concentration (inhibits trypsin and protonates tryptic peptides (charged for electrospray)).
- Desalt mixture by HPLC with a Microm Bioresources C8 peptide macrotrap (3x8mm), 20µg maximum capacity, and lyophilize (optional method, but the solution will need to be desalted by some method before LC-MS analysis).
- Leave lyophilized peptides dry until ready for analysis then resuspend lyophilized peptides in 0.2% formic acid.