Trypsin On-bead Digestion Protocol

Note:

• Do not reduce/alkylate Co-IP beads as antibodies could be less resistant to trypsin after breaking the disulfide bonds, resulting in a significantly increased PSM of the antibody and thus impacting the identification of other proteins. (https://www.nature.com/articles/nprot.2016.020 )

• Discuss with sample submitter whether to reduce/alkylate for other kinds of beads (for example streptavidin magnetic beads).

• Adjust volumes and amount of trypsin based on the amount of the beads.

• The typical binding capacity of the MagReSyn streptavidin magnetic beads (10 mg/mL stock) is 250 μg IgG per 1 mg of bead.

1. Bead wash: optional (when the beads are too diluted or the storage buffer is not Ammonium Bicarbonate)

• Discard the original buffer.

• Wash the beads with 100 μL of 50 mM Ammonium Bicarbonate (ABC).

• Discard the ABC buffer.

• Add 50 μL of 50 mM ABC to the beads.

2. Reduction and Alkylation: optional (discuss with sample submitter to determine if this step is desired)

• For beads resuspended in 50 μL ABC, add 2.5 μL 100 mM TCEP (final 5 mM). Shake at room temperature or 25 °C for 30 minutes.

• Add 2 μL 400 mM Chloroacetamide (CAM, final 15 mM), incubate at room temperature for 30 minutes in dark.

• Add 3 μL 100 mM TCEP to quench the alkylation reaction, shake at room temperature or 25 °C for 10 minutes.

3. Add 2-5 μL 100 ng/μL trypsin, digest overnight at 37 °C with shaking at 900 rpm.

4. Add 3 μL 10% TFA to acidify the sample.

5. Transfer the supernatant to a new 0.65 mL tube and dry down the sample in speedvac.

6. Proceed to zip-tip desalting protocol and dry down the desalted peptide in speedvac.

7. Resuspend the dried sample in 7 μL 0.1% Formic Acid (FA) and inject 5 μL on LC-MS instrument.