SP 4 Protocol
This protocol is adapted from: Solvent Precipitation SP3 (SP4) Enhances Recovery for Proteomics Sample Preparation without Magnetic Beads, https://doi.org/10.1021/acs.analchem.1c04200, Anal. Chem. 2022, 94, 10320−10328.
The ideal samples for this protocol are 50 µl in volume (in a lysis buffer of the researcher’s choice) and at a concentration of 1 µg/µl. Adjust the reagent volumes below if you are not starting with this volume or concentration.
Samples should be submitted in 1.5 ml Eppendorf tubes (Protein Lo-bind preferred).
Samples should be submitted on dry ice and stored at -80 ºC until they can be processed.
Thaw samples by placing them on a Thermomixer at 25 ºC for 10 minutes at 800 rpm.
1. Denaturation
Add 150 µl of 8 M urea (suggested reagent is Sigma, catalog number U4883).
Samples are now 200 µl in volume and the urea concentration is 6 M.
Place on a Thermomixer at 25 ºC for 10 minutes at 800 rpm.
2. Reduction
Add 11 µl of aqueous 100 mM TCEP to each sample to give an effective final concentration of approx. 5 mM.
Place on a Thermomixer at 25 ºC for 20 minutes at 800 rpm.
3. Alkylation
Add 6 µl of aqueous 400 mM CAM (Aldrich, 22790, 2-chloroacetamide) to each sample to give an effective final concentration of approx. 10 mM.
Place on a Thermomixer at 25 ºC for 20 minutes at 800 rpm in the dark.
There are stock solutions of aqueous 100 mM TCEP and 400 mM CAM in the -20 ºC freezer.
4. Protein precipitation (overnight)
Remove the bottle of acetonitrile from the -20 ºC freezer.
Add 1,000 µl of ice-cold acetonitrile to each sample.
Do not aspirate or mix when the acetonitrile is added but do gently vortex to mix for 5 seconds once all samples have had acetonitrile added to them.
Store in the -20 ºC freezer overnight.
The following day, remove samples from the freezer.
Immediately centrifuge for 5 minutes at 10,000 ×g with the tube hinge facing out.
Using a 1 ml pipette, remove most of the supernatant without disturbing the pellet.
Leave approx. 10 µl of liquid at the bottom of the tube.
5. Pellet washing
Make 1 ml of 80 % ethanol by adding 200 µl water to 800 µl of ethanol.
Add 80 µl of 80 % ethanol to each sample by pipetting carefully down the opposite side to the hinge/pellet to avoid disturbing it. Do not vortex.
Centrifuge for 2 minutes at 10,000 ×g.
Using a gel-loader pipette tip, remove 85 µl of the supernatant wash to leave approx. 5 µl of liquid.
Repeat the wash two further times for a total of three washes, remove about 80 uL of the supernatant each time to leave approx. 5 uL of liquid.
Air dry the pellet.
6. Digestion
Add 50 µl of aqueous 50 mM AMBIC (Sigma Aldrich, A6141, ammonium bicarbonate) digestion buffer to sample to solubilize pellets.
Remove a “1×T” trypsin vial from the freezer and thaw. This is 20 µl of 100 ng/µl trypsin in acetic acid.
Add 10 µl (approx. 1 µg so a 1:50 ratio of trypsin to protein) of trypsin to each sample.
Incubate in a Thermomixer at 600 rpm at 37 °C overnight.
7. Peptide recovery
The next day, centrifuge each sample for 2 minutes at 1,400 ×g.
Transfer the peptide solution to a new 0.65 ml tube.
Add 20 µl of room-temperature acetonitrile.
Dry by vacuum centrifugation for 60 minutes.
7. Peptide Desalting
Dried peptide samples can be res-suspended in 20 µl of solvent A ready for the Ziptip protocol.
After Ziptipping, dry by vacuum centrifugation for 60 minutes.
Re-suspend in 7 µl of solvent A and inject 5 µl for LC-MS analysis.