TITLE: Manual SP3 digestion and clean-up of protein lysates
AUTHORS: ronan.ocualain

[DESCRIPTION]
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 10 mg, 50 mg, and  100 mg volumes, store at 4 °C until further use.  

[GUIDELINES]
pH: SP3 works in the pH range of pH 7.0 to pH 8.5.
Protein samples should be ultrasonicated to remove nucleic acid. DNA if in sample will coat the SP3 beads, causing their aggregation, and is best avoided. If ultrasonication is not available, use Benzonase to shear DNA.
Bead concentration during binding: A bead to protein ratio of  5:1 to 10:1 is recommended. For example, if processing 20 µg of protein, add 
Sample concentration: The binding capacity of SP3 beads provides a flexible clean-up format across a range of protein and peptide concentrations (10 μg/mL to 5 mg/mL), as long as the concentration of beads is adjusted as described above.
Sample handling: The use of 200 μL gel loading pipette tips for sample aspiration is recommended.  Care should be taken to gently aspirate supernatant to avoid dislodging protein or peptide-bound beads from the magnet. 
QC: Aliquots of the load, flow-through, washes, and bound bead: sample may be processed by SDS PAGE and visualised with Colloidal Coomassie or SIlver staining to determine binding efficiency of the sample to beads, and if wash steps are sufficient.

[STEPS]
SECTION: SP3 bead preparation
1. Briefly vortex the 1:1 bead mixture and place the tube on a magnetic stand for two minutes to collect the beads. 

SECTION: SP3 protein clean-up
6. Check the pH of the sample is in the range of pH 7.0 to pH 8.5 for optimal binding by measuring an aliquot on pH paper.

SECTION: SP3 protein clean-up
7. Add washed beads (prepared as above) to the samples in a ratio of 5-10 µg of beads to 1 µg of protein and briefly vortex.

SECTION: SP3 protein clean-up
8. 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.

SECTION: SP3 protein clean-up
9. Vortex the samples to mix but ensure that beads are not stuck on the sides of the tube.
 

SECTION: SP3 protein clean-up
10. Incubate the samples on a room temperature mixer platform for 10 minat 1000 rpm

SECTION: SP3 protein clean-up
11. Remove the samples from the mixer, centrifuge them for 2 s and place them on the magnetic stand for 2 min.

SECTION: SP3 protein clean-up
12. Transfer the supernatants to a clean Sarstedt tube. This is the "flow-through" fraction.
SECTION: SP3 protein clean-up
14. Centrifuge the samples  briefly for two seconds on a mini centrifuge and place back on the magnetic stand for 2 min	

SECTION: SP3 protein clean-up
15. Remove the supernatants and save in a separate Sarstedt tubes. Label these wash 01.
SECTION: SP3 protein clean-up
16. 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 2 min 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). 

SECTION: SP3 protein clean-up
17. After the final wash, air dry the beads for 30 s to remove as much ethanol as possible.

SECTION: SP3 digestion
18. Resuspend the beads in 25 µL of 100 millimolar (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). 
SECTION: SP3 digestion
20. Add Promega™ sequencing grade trypsin in a 1:20 enzyme-to substrate ratio to each sample, and digest at 37 °C 2 min  on an Eppendorf thermomixer.

SECTION: SP3 digestion
21. After 16 to 18 hours, add the tubes to a magnetic stand for 2 min . Carefully remove the supernatant, and transfer to a clean, labelled tube. Add an additional 60 µL volume of 100 millimolar (mM) ammonium bicarbonate buffer to the beads, briefly vortex, and allow to stand for  2 min on a magnetic rack. Carefully transfer the supernatant to the same labelled sample tube, to create a pooled sample. 

SECTION: SP3 bead preparation
2. Add ultrapure water at a volume corresponding to 5 to 10 times the initial volume of mixed beads.
Vortex the beads for 10 s and place on a magnetic stand for 2 min to collect the beads. Carefully aspirate and discard the wash buffer with a gel loading tip.
                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                    
SECTION: SP3 bead preparation
3. 
Repeat the wash steps a further two times.

SECTION: SP3 bead preparation
4. Resuspend the beads with ultrapure water at a final concentration of 10 µg/µL.

SECTION: SP3 protein clean-up
13. Wash the beads by adding a volume of 80 % (v/v) ethanol corresponding to at least twice the initial sample volume and vortex for 30 s

SECTION: SP3 digestion
19. Do not vortex the beads at this stage. Instead, place the tubes on a floating rack and sonicate in a water batch for 2 min to resuspend them.
SECTION: SP3 digestion
22. Centrifuge the peptide for 10 minutes at 14000 x g, and proceed to R3 desalting (see https://www.protocols.io/view/96-well-plate-r3-desalt-and-clean-up-protocol-for-dm6gpbnqdlzp/v1 )

SECTION: SP3 bead preparation
5. Washed beads may be stored at 4 °C for up to one month.