On-bead Trypsin Digest (based on protocol 259 from A. C. Gingras lab, modified)

• Follow your standard IP protocol
• After the last lysis buffer wash, perform additional 2 washes on ice with 1 mL of ice-cold 20mM TrisHCl pH8 + 2mM CaCl2 (this should not have protease inhibitors!).
• Remove a small portion of the beads for WB or silver stain analysis.
• Centrifuge beads quickly and remove the last drops of liquid with a fine pipette.

* OK to freeze beads here and continue later

• Resuspend the liquid-free beads in 9 uL of 20 mM Tris-HCl pH 8.0. Add 0.4 uL of 100 mM DTT and incubate at RT for 30 min with agitation (e.g. slow Vortex).
• Add 0.6 uL of 100 mM iodoacetamide and incubate at RT for 10 min with agitation.
• Add 500ng of trypsin (Sigma Trypsin Singles, T7575) and incubate at 37°C on a rotator overnight (~12 hrs).
• After this first incubation, magnetize the sample and transfer the supernatant to a fresh tube.
• Add additional 500ng of trypsin (in 5uL of 20mM Tris-HCl pH8) and incubate at 37°C for 3-4 hours with gentle agitation.
• Stop the digest by adding formic acid to a final concentration of 2% (e.g. use a 50% formic acid stock solution).
• Store sample at -80°C.
• ZipTip sample before MS analysis