Manual SP3 digestion and clean-up of protein lysates
ronan o'cualain
Abstract
SP3 bead preparation of samples for MS analysis
Compatible with a wide range of buffer and detergent types.
Before start
Bead preparation:
Both types of Cytiva Carboxylate SpeedBeads are shipped at 50 mg/mL concentration (5% solids) in water with 0.05% sodium azide. It is a good idea to combine the beads and aliquot them for long term storage at 4°C . Preparing aliquots of stock beads avoids excess handling of the main bottles and minimizes the risk of contamination.
To do this: Let both stock beads equilibrate to room temperature for 30 minutes. If the beads have settled during storage they should be resuspended by inversion or gentle vortexing until no solid bead mass is visible at the bottom of the bottle. Combine both types of beads into a 1:1 ratio, and aliquot into 10mg, 50mg, and 100mg volumes, store at 4°C until further use.
Steps
SP3 bead preparation
Briefly vortex the 1:1 bead mixture and place the tube on a magnetic stand for two minutes to collect the beads.
Add ultrapure water at a volume corresponding to 5 to 10 times the initial volume of mixed beads.
Vortex the beads for 0h 0m 10s and place on a magnetic stand for 0h 2m 0s to collect the beads. Carefully aspirate and discard the wash buffer with a gel loading tip.
Repeat the wash steps a further two times.
Resuspend the beads with ultrapure water at a final concentration of 10µg/µL.
Washed beads may be stored at 4°C for up to one month.
SP3 protein clean-up
Check the pH of the sample is in the range of 7.0 to 8.5 for optimal binding by measuring an aliquot on pH paper.
Add washed beads (prepared as above) to the samples in a ratio of 5-10µg of beads to 1µg of protein and briefly vortex.
Immediately add a volume of 100% (v/v) ethanol to the samples to obtain a 50% final concentration to initiate protein binding to the beads.
Vortex the samples to mix but ensure that beads are not stuck on the sides of the tube.
Incubate the samples on a room temperature mixer platform for 0h 10m 0sat 1000rpm,0h 0m 0s
Remove the samples from the mixer, centrifuge them for 0h 0m 2s and place them on the magnetic stand for 0h 2m 0s.
Transfer the supernatants to a clean Sarstedt tube. This is the "flow-through" fraction.
Wash the beads by adding a volume of 80% (v/v) ethanol corresponding to at least twice the initial sample volume and vortex for 0h 0m 30s
Centrifuge the samples briefly for two seconds on a mini centrifuge and place back on the magnetic stand for 0h 2m 0s
Remove the supernatants and save in a separate Sarstedt tubes. Label these wash 01.
Repeat the wash steps a further three times, and transfer the supernatants to clean Sarstedt tubes, labelled wash 02 , wash 03 , and wash 04.
For the final wash (wash 04) - perform this by transferring the resuspended beads in 80% (v/v) ethanol to a new, labelled tube. Stand for 0h 2m 0s on a magnetic rack, and remove the supernatant, the beads are now ready for digestion.
(This is a critical step, because residual detergent on the sides of the tube and even beads may be transferred to the downstream steps).
After the final wash, air dry the beads for 0h 0m 30s to remove as much ethanol as possible.
SP3 digestion
Resuspend the beads in 25µL of 100millimolar (mM) ammonium bicarbonate buffer
(This is a critical step, because residual detergent on the sides of the tube and even beads may be transferred to the downstream steps).
Do not vortex the beads at this stage. Instead, place the tubes on a floating rack and sonicate in a water batch for 0h 2m 0s to resuspend them.
Add Promega™ sequencing grade trypsin in a 1:20 enzyme-to substrate ratio to each sample, and digest at 37°C 0h 2m 0s on an Eppendorf thermomixer.
After 16 to 18 hours, add the tubes to a magnetic stand for 0h 2m 0s . Carefully remove the supernatant, and transfer to a clean, labelled tube. Add an additional 60µL volume of 100millimolar (mM) ammonium bicarbonate buffer to the beads, briefly vortex, and allow to stand for 0h 2m 0s on a magnetic rack. Carefully transfer the supernatant to the same labelled sample tube, to create a pooled sample.
Centrifuge the peptide for 10 minutes at 14000x g,0h 0m 0s, and proceed to R3 desalting (see https://www.protocols.io/view/96-well-plate-r3-desalt-and-clean-up-protocol-for-dm6gpbnqdlzp/v1 )
