This protocol is utilized in the procedures of the following manuscripts, and has been optimized for use with ThiopropylSepharose 6B (GE Healthcare) resin:
The procedure is scaled for approximately 20 uL of TPS resin. Scale volumes appropriately if using more or less resin.
After incubation to capture free thiols:
- 1. Wash resin 3x with 1 mL 50mM Ammonium Bicarbonate , pH 8 (AmBic), removing the supernatant each time with spinning at 12000 rcf in a micro swinging‐bucket centrifuge for 2 minutes.
- Add 100 uL of 0.25% ALS‐1 AmBic.
- Add 5 uL of 0.1 ug/uL Promega Sequencing Grade Trypsin (0.5 ug).
- Incubate overnight at 37C, 900 rpm on Thermomixer R. The speed may need to be increased depending on the quantity of resin and volume of liquid. The goal is to keep the resin suspended during trypsinization.
- Spin at 12000 rcf in a micro swinging‐bucket centrifuge and remove supernatant in the morning and set aside. Supernatant may be analyzed directly by LC‐MS/MS in cases where the non‐Cys‐containing peptides from the bound proteins are of interest.
- For all of the following wash steps, add 250 uL of listed buffer, vortex for a few seconds, spin at 12000 rcf in the swinging bucket centrifuge for 2 minutes, and then discard supernatant before next wash, but use caution not to pipette too close to resin bed. A gel‐loader tip with vacuum suction can be an alternative way to decant the supernatants (again, be careful not to suction up the resin!).
- a. Wash resin 3x with HENS buffer (250 mM HEPES pH 7.7, 1mM EDTA, 0.1mM Neocuproine, 1%SDS).
- Wash resin 3x with 50 mM AmBic, pH 8.
- Wash resin 3x with 80% ACN/0.1% TFA.
- Wash resin 3x with 50% MeOH.
- Wash resin 3x with 50 mM AmBic, pH8.
- Elute thiol‐containing peptides by adding 100 uL of 20 mM dithiothreitol in AmBic and incubate at 37C on Thermomixer R (900 rpm) for 45 minutes.
- Spin to pellet the resin, then pipette off peptide‐containing supernatant into low bind Epi tube.
- Rinse resin with 20 uL of 50 mM AmBic, vortex, spin, and add supernatant from wash to peptides in step 9.
- Alkylate with 100 uL of 80 mM iodoacetamide by incubation at room temperature in the dark for 30 min.
- Reduce the volume of the samples in speed vac to approximately 20 uL, then perform a C18 Zip Tip (Millipore) cleanup of the peptides, following the manufacturers protocol.
- Dry Zip Tip eluent and resuspend in normal LC/MS buffer. If protocol was performed properly, the peptides in the SNO‐RAC or Acyl‐RAC sample should be 95+% Cys‐containing peptides.