TITLE: SP3 (Single-Pot, Solid-Phase, Sample-Preperation) Protein Extraction for Dental Calculus
AUTHORS: Shevan Wilkin, Richard Hagan, Sandra Hebestreit, Madeleine Bleasdale, Ayushi Nayak, Li Tang, Traci N Billings, Nicole Boivin, Kristine Richter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Dental calculus from archaeoloigcal samples is now commonly extracted for palaeoproteomic analysis. This protocol combines an EDTA based pretreatment and a SP3 (single-pot, solid-phase-enhanced) protein extraction to prepare samples for LC-MS/MS analysis. This protocol is for moderate to well preserved archaeological calculus samples with a starting weight of 5-15 mg of calculus.  We recommend batch sizes of 2-10 calculus samples, 1 extraction control (we use a powdered bone sample from an achaeological sheep bone as this should not be modern material), and 1 extraction blank.  For the first time doing the protocol in a lab, we recommend using flanking blanks to identify any contaminants as from handling or from solutions.</div><div class = "text-block">This protocol is based upon the original SP3 protocol published in Hughes et al. (2014) and the developments for palaeoproteomics of bone (Cleland et al., 2018).  When using please cite the following three publications the protocol is based upon as well as the DOI for this protocol.</div><div class = "text-block"><span>Hughes, C. S., Foehr, S., Garfield, D. A., Furlong, E. E., Steinmetz, L. M., & Krijgsveld, J. (2014). Ultrasensitive proteome analysis using paramagnetic bead technology. </span><span style = "font-style:italic;">Molecular Systems Biology</span><span>, </span><span style = "font-style:italic;">10</span><span>, 757. https://doi.org/10.15252/msb.20145625</span></div><div class = "text-block"><span>Hughes, C. S., Moggridge, S., Müller, T., Sorensen, P. H., Morin, G. B., & Krijgsveld, J. (2019). Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. </span><span style = "font-style:italic;">Nature Protocols</span><span>, </span><span style = "font-style:italic;">14</span><span>(1), 68–85. https://doi.org/10.1038/s41596-018-0082-x</span></div><div class = "text-block"><span>Cleland, T. P. (2018). Human Bone Paleoproteomics Utilizing the Single-Pot, Solid-Phase-Enhanced Sample Preparation Method to Maximize Detected Proteins and Reduce Humics. </span><span style = "font-style:italic;">Journal of Proteome Research</span><span>, </span><span style = "font-style:italic;">17</span><span>(11), 3976–3983. https://doi.org/10.1021/acs.jproteome.8b00637</span></div></div>

[STEPS]
?. [Reagent Preparation]
General InformationWe recommend to use combusted glassware for buffers and solutions. Solutions should be prepared under a dedicated dead air hood for reagent preparation.TFA should be handled only under a fume hood.Prepare the Guanidine Hydrochloride solution last! All bottle and tubes containing stock solutions shall be labelled with name of the chemical, concentration and the date.For buffers and solution preparation we recommend the usage of UltraPure DNase/RNase-Free Distilled Water (see materials list, in this protocol referred to as "molecular grade water").
?. [Reagent Preparation]
Bead Solution ( ; Shelf life: 1 month at  , mix before use)
4 °C
?. [Reagent Preparation]
Carefully invert the bottles of bead stock solutions and knock (!) them firmly on the counter to gain an equal distribution of the beads in the stock solution.
Stock concentration of the bead stock solutions: 50 mg/ml.Beads tend to be very sticky! Make sure you can visibly see that the beads are no longer stuck to the walls of the bottle before pipetting.
?. [Reagent Preparation]
Pipette   of the hydrophobic and hydrophilic beads each into one microcentrifuge tube (combine them).
200 µl
?. [Reagent Preparation]
Place the tube on the magnetic rack, remove the stock supernatant and discard.
?. [Reagent Preparation]
Replace with   molecular grade water and resuspend the beads through up and down pipetting (first wash).
1000 µl
?. [Reagent Preparation]
Repeat the wash 2x.
?. [Reagent Preparation]
After the final wash, add   molecular grade water to the beads and resuspend to mix.
1000 µl
Bead working concentration is now
?. [Reagent Preparation]
Reduction and Alkylation solution (Shelf life of weighed out powder: 6 months at )
4 °C
?. [Reagent Preparation]
Reduction and alkylation solution is   of   CAA (2-Chloracetamide),   TCEP (Tris(2-carboxyethyl)phosphine) prepared during extraction.To facilitate this, weigh out:  of TCEP into a 1.5 or 2.0 ml tube  of CAA into a 1.5 ml black tube as it is slightly light sensitive
1 mL
28.66 mg
9.35 mg
?. [Reagent Preparation]
Guanidine Hydrochloride (GuHCl) (Shelf life: 3 months at  )
4 °C
Prepare the GuHCl soltution last .
Never mix or clean up GuHCl with bleach (Sodium hypochloride). This will create chlorine gas which is highly toxic and would necessitate the immediate evacuation of your lab. After preparing the GuHCl solution, wipe down all surfaces with ultrapure water before cleaning the surfaces with bleach.
?. [Reagent Preparation]
Weigh out   GuHCl powder into a 50 ml tube.
28.66 g
?. [Reagent Preparation]
Aliquot out around   of water into a 50 ml tube.
40 mL
?. [Reagent Preparation]
Now mix the GuHCl with   molecular grade water and check the volume.
25 mL
?. [Reagent Preparation]
Fill up to 50 ml with molecular grade water and transfer the solution to a 50 ml glass bottle for long-term storage.
?. [Reagent Preparation]
and  Ethanol (Shelf Life: 1 month at  )
4 °C
?. [Reagent Preparation]
Ethanol: Aliquot out   of HPLC grade Ethanol into a 50 ml bottle. Ethanol: Add   of HPLC grade Ethanol into a 50 ml bottle. Add   of molecular grade water.
50 mL
40 mL
10 mL
?. [Reagent Preparation]
Ammonium bicarbonate (AmBic,  , Shelf life: 6 months at  )
4 °C
?. [Reagent Preparation]
Weight out   of AmBic powder.
395 mg
?. [Reagent Preparation]
Dissolve AmBiC powder in   molecular grade water and mix by shaking gently.
50 mL
?. [Reagent Preparation]
Trifluoroacetic acid (TFA) (Shelf Life: 3 months at  )
0 Room temperature
Triflouroacetic acid (TFA) is very hazardous (corrosive)! Handling should only be performed under a fume hood.Prepare the solution in a brown glass bottle as TFA is light sensitive. Always pipette water first, then add the acid slowly! Repeated used of plastic pipettes in TFA stock solutions causes plastics to build up in the TFA stock solution and can interfere with mass spectrometry, therefore ALWAYS use a glass pipette/syringe for TFA stock solution. Working solutions with TFA for this protocol are 5% and can be handled outside of a fume hood and with plastic pipettes.
?. [Reagent Preparation]
Dilute   TFA stock solution using a glass syringe/pipette (no plastic) in   molecular grade water and mix by shaking carefully.
1 mL
19 mL
?. [Reagent Preparation]
MeOH (Shelf life: 1 month at  )
4 °C
?. [Reagent Preparation]
Aliquot out  analysis grade MeOH into a 50 ml bottle.
50 mL
?. [Reagent Preparation]
Acetonitrile (ACN),  Trifluoracetic acid (TFA) (Shelf life: 3 Months at  )
4 °C
Triflouroacetic acid (TFA) is very hazardous (corrosive)! Handling should only be performed under a fume hood.Repeated used of plastic pipettes in TFA stock solutions causes plastics to build up in the TFA stock solution and can interfere with mass spectrometry therefore ALWAYS use the glass pipette for the TFA.
?. [Reagent Preparation]
First pour roughly   molecular grade water into a 100 ml glass bottle.
80 mL
?. [Reagent Preparation]
Add   ACN by pipetting and fill up to  with molecular grade water.
3 mL
100 mL
?. [Reagent Preparation]
Add   TFA with a glass syringe/pipette.
100 µl
?. [Reagent Preparation]
Acetonitrile (ACN),  TFA (Shelf life: 3 Months at  )
4 °C
Triflouroacetic acid (TFA) is very hazardous (corrosive)! Handling should only be performed under a fume hood.Repeated used of plastic pipettes in TFA stock solutions causes plastics to build up in the TFA stock solution and can interfere with mass spectrometry therefore ALWAYS use the glass pipette for the TFA.
?. [Reagent Preparation]
First pour   molecular grade water in a 50 ml glass bottle.
20 mL
?. [Reagent Preparation]
Add   ACN.
30 mL
?. [Reagent Preparation]
Add  TFA with a glass syringe/pipette.
50 µl
?. [Reagent Preparation]
Trypsin solution (Stable once resuspended for 1 month at  )
-20 °C
?. [Reagent Preparation]
Freeze dried Trypsin ( , stored at  ) is resuspended in  of trypsin resuspension buffer (included in Trypsin Kit, see materials list). The final concentration is now  .
20 µg
-20 °C
100 µl
?. [Reagent Preparation]
Transfer the solution into a labelled 0.5 ml tube and store at  .
-20 °C
?. [Sample Preparation]
Weigh out the samples and positive control
General Information and preparation:All steps should take place in a dedicated sample preparation hood.Wipe down all surfaces (1. 5% (v/v) bleach, 2. ultrapure water, 3. 70% (v/v) Ethanol).Alliquot out the reagents you will need (EDTA).
?. [Sample Preparation]
Weigh out -    calculus in a microcentrifuge tube and record the exact weight.
5 mg
10 mg
Calculus needs to be removed from the teeth using a dental scaler prior to analysis.
?. [Sample Preparation]
Weigh out -  of positive control.
2 mg
3 mg
We use a powdered archeological sheep bone as positive control. Do not use modern material.
?. [Sample Preparation]
DemineralizationAdd  of  EDTA (from your aliquot) to each sample and place on the rotator.
500 µl
Do not forget to prepare an extraction blank tube with EDTA only at this step.
?. [Sample Preparation]
Samples (including the positive control and extraction blank) should be left on the rotator for 4-7 days at  until the calculus (and bone) samples look visibly demineralised. Your blank should remain clear.
0 Room temperature
The demineralized calculus can range from being completely invisible to feathery bits floating around in the EDTA. There should be no mineralized chunks remaining. If there are, leave the samples to further demineralize for an extra couple of days.
If the room temperature is > 30 °C or fluxuates greatly, demineralization can be performed at   or   in an incubator (see materials list).
If not immediately proceeding to Day 1 of SP3 (see step 14), samples can be stored in the freezer at  . Thaw samples completely after freezing before continuing.
?. [SP3 Protocol Day 1]
General Information and preperation:!Start this section only when your samples are adequately demineralized!All preparations and pretreatment steps should take place in a dedicated extraction hood.Wipe down all surfaces (1.Bleach, 2. ultrapure water, 3. 70% (v/v) Ethanol).Alliquot out the reagents that you need (6 M GuHCl, 100 mM AmBiC, molecular gerade water, 100% (v/v) Ethanol, 80% (v/v) Ethanol)Check if you have prepared Trypsin solution (0.2 µg/µl), working bead solution (20 µg/µl), weighed out CAA/TCEP , StageTips, and clean magnetic racks.
?. [SP3 Protocol Day 1]
Turn the Thermoshaker to  in preparation for the heating step.
99 °C
?. [SP3 Protocol Day 1]
Remove all samples from the rotator and centrifuge  (or max speed) to separate the supernatant from the pellet.
Centrifuge: 20.000 34, 10 min
?. [SP3 Protocol Day 1]
During centrifugation, label a new set of microcentrifuge tubes.
These tubes are to collect the EDTA supernatant (see Step 19) to keep as backup. Use the suffix "EDTA sup" for labeling.
?. [SP3 Protocol Day 1]
Prepare the  CAA/ TCEP solution.
?. [SP3 Protocol Day 1]
Add  molecular grade water to the TCEP preweighed tube and vortex until dissolved.
1 mL
?. [SP3 Protocol Day 1]
Transfer the TCEP solution to a black CAA preweighed tube and vortex for .
?. [SP3 Protocol Day 1]
Remove  of the original samples’ supernatant and transfer to the tubes labelled with “EDTA Sup.” Store at  for backup. You should have  and the pellet remaining in the original sample tube.
400 µl
-20 °C
100 µl
?. [SP3 Protocol Day 1]
Denaturing and LysisAdd  of the  GuHCl to each sample and resuspend to mix.
200 µl
You should see most, if not all, of the pellet disappear. If you have humics, such as dirt or other non-mineralized materials in your sample, these may still be in the pellet and they will be removed in later rinse steps.
?. [SP3 Protocol Day 1]
Briefly centrifuge the samples to get the liquid to the bottom of the tube.
?. [SP3 Protocol Day 1]
Reduction and AlkylationAdd  of the prepared  CAA/ TCEP solution to each tube
30 µl
Final working concentration should be   CAA/   TCEP. Adjust if you use a different starting volume.
?. [SP3 Protocol Day 1]
Vortex each sample quickly (~ 2 sec) and place the samples in the Thermoshaker for  .
Centrifuge: 0 33, 10 min, 99 10
?. [SP3 Protocol Day 1]
While waiting for your samples on the Thermoshaker, label a new set of microcentrifuge tubes.
These tubes are to collect a further supernatant (see Step 32) to keep as backup. Use the suffix "SP3 sup" for labeling.
?. [SP3 Protocol Day 1]
After 10 min of incubation, remove your tubes and give your samples approximately  to cool and turn the Thermoshaker to .
24 °C
If necessary, you can speed up the cooling process of the Thermoshaker with prepared microcentrifuge tubes of ice.
?. [SP3 Protocol Day 1]
Briefly centrifuge the samples to get the liquid to the bottom of the tube.
?. [SP3 Protocol Day 1]
Protein PurificationAdd  of the working bead solution () to each tube to begin SP3 clean-up and mix by gentle pipetting.
20 µl
Ensure complete homogenization of the SP3 beads in the solution. Improper mixing will reduce the protein recovery.
Final bead concentration at this point is  .  It is recommended that during binding (after the addition of Ethanol) the ratio of beads to protein should be 1:10, but no lower than   . Adjust the amount of bead solution used if you use a different volume or know you have an extremely high protein concentration.
Once the beads are added, the sample cannot be stored or frozen until after the beads are removed.
?. [SP3 Protocol Day 1]
DigestionRemove the tubes from the magnetic rack and add  of  AmBic solution to each sample.
75 µl
Use the tip of a pipette to push any beads down into the AmBic solution, but do not actually pipette/resuspend the beads at this point. Also do not shake, flick or invert the tube.
?. [SP3 Protocol Day 2]
AcidificationRemove the samples from the Thermoshaker and centrifuge them briefly.
?. [SP3 Protocol Day 2]
Place the tubes on the magnetic rack and wait until all beads have migrated to the wall.
?. [SP3 Protocol Day 2]
As you wait, Label a new set of microcentrifuge tubes with your sample IDs.
These tubes will not be retained after use.
?. [SP3 Protocol Day 2]
Transfer the entire sample supernatants to the labelled tubes and discard the tubes with the remaining beads.
?. [SP3 Protocol Day 2]
Acidify the samples by adding  of TFA to bring the pH below .
10 µl
?. [SP3 Protocol Day 2]
Quickly vortex the sample and test the pH using - of sample on the pH strips.
1 µl
4 µl
If   does not lower the pH enough, add an additional   of   TFA until the desired pH is reached (It is essential to acidify the samples in order to stop the tryptic digestion and to enable the peptides to adhere to the stage tip C18 material.Record the total volume of   TFA added to each sample and the resulting pH to keep track of any anomalies.
?. [SP3 Protocol Day 2]
Peptide Purification/StageTippingPrepare 2.0 ml tubes by placing stage tip holders into them to allow the StageTips to be inserted. Label a set of stage tips with your sample IDs.
It is necessary to clean the StageTips before loading the samples. Since the waste from cleaning the StageTips will be discarded prior to peptide collection, it is not necessary to label this set of 2.0 ml tubes with your sample IDs.
Be careful when handling the stage tips. Discard any stage tips if the filters are not properly situated in the tips or are disrupted during handling.
?. [SP3 Protocol Day 2]
Prepare a second set of 2.0 ml tubes with your samples IDs.
These tubes are to collect the the load and wash flow-throughs during peptide purification (see Step 32) to keep as backup. Use the suffix "L+W" for labeling.
?. [SP3 Protocol Day 2]
Add  of Methanol (MeOH) to the StageTip. Centrifuge at .
150 µl
Centrifuge: 2000 34, 1 min
Check your StageTips after every centrifugation step to see if all the liquid went through and that the and that the filter is still properly situated in the StageTip. If the filter has become dislodged, discard the StageTip and prepare a new one.
?. [SP3 Protocol Day 2]
To equilibrate the StageTips, add  of ACN, TFA and centrifuge at .
150 µl
Centrifuge: 2000 34, 1 min
Check your StageTips after centrifugation to see if all the liquid went through and that the StageTip is still intact.
?. [SP3 Protocol Day 2]
Carefully transfer the StageTip in its holder to the 2.0 ml tube labelled “Load/Wash” and discard the waste accumulated in the tubes.
?. [SP3 Protocol Day 2]
Repeat the above step (add  of  ACN,  TFA and centrifuge at).
150 µl
Centrifuge: 2000 34, 1 min
Check your StageTips after centrifugation to see if all the liquid went through and that the StageTip is still intact.
?. [SP3 Protocol Day 2]
Centrifuge the tubes containing your acidified samples for a short spin.
If samples contain particles or undigested parts of the pellet at this point, you can spin longer or at higher speeds in order to avoid transferring accumulated particles to the StageTip as these could clog it.
?. [SP3 Protocol Day 2]
Place the acidified sample tubes back on the magnetic rack to make sure no remaining beads are transferred to the StageTip.
Avoid transferring any beads or any particulates to the StageTip as these could clog it.
?. [SP3 Protocol Day 2]
Centrifuge: 2000 34, 3 min
Check each StageTip to see if all the liquid has completely passed through. If not, continue centrifugation.
?. [SP3 Protocol Day 2]
Add  of  ACN,  TFA to the StageTip and centrifuge at .
150 µl
Centrifuge: 2000 34, 2 min
Check each StageTip to see if all the liquid has completely passed through. If not, continue centrifugation.
?. [SP3 Protocol Day 2]
Repeat the above step (add  of  ACN,  TFA to the StageTip and centrifuge at  for ).
150 µl
Centrifuge: 2000 34
Check each Stage tip to see if the liquid has completely passed through! If not, continue centrifugation until all samples have passed the tip.
?. [SP3 Protocol Day 2]
Store the “Load/Wash” tubes at  as backup.
-20 °C
?. [SP3 Protocol Day 2]
Store the stage tips containing the peptides until they undergo elution for mass-spetrometric analysis.
-20 °C
If eluting: Transfer StageTip with the holder to a new tube and add    ACN,   TFA to the StageTip and centrifuge at  .
?. [SP3 Protocol Day 1]
Add  of  Ethanol to your sample and briefly shake the tube by hand to mix.
350 µl
Avoid excessive shaking and do not flick or invert the tube. Both can cause protein loss due to beads sticking to the upper areas of the tube. Complete homogenization is not essential as it will be achieved during the incubation on the Thermoshaker.
Volume of   Ethanol should be the same as the volume of solution in the tube. Adjust if you use a different volume.
?. [SP3 Protocol Day 1]
Incubate your samples on the Thermoshaker at  .
Centrifuge: 1000 33, 5 min, 24 10
?. [SP3 Protocol Day 1]
Place the tubes on a magnetic rack and wait for the beads to migrate to the wall (approx. ).
?. [SP3 Protocol Day 1]
Remove the sample/Ethanol solution, without  disturbing the beads, and place in the tubes labelled “SP3 Sup”. Store at  as a backup.
-20 °C
Set the Thermoshaker to   in preparation for the tryptic digestion (see step 28).
?. [SP3 Protocol Day 1]
Remove the tubes from the magnetic rack and add of   Ethanol and resuspend the beads in the solution to rinse.
200 µl
Aggressive or excessive pipetting during washing can result in protein loss.
?. [SP3 Protocol Day 1]
Put the tubes back on the magnetic rack and wait for the beads to migrate to the magnetic wall. Remove and discard the supernatant without disturbing the beads.
?. [SP3 Protocol Day 1]
Repeat  Ethanol wash two additional times for a total of three washes ( see step 27.5 and 27.6).
Calculus samples can be extremely “dirty” and the beads may become sticky. If the beads stick to the walls, gently push them with a pipette tip back into the liquid so they can fully migrate to the tube wall which is against the magnetic rack. Each rinse should reduce this “stickiness” and the beads should easily migrate to the wall by the end. There is no harm in performing an extra rinse if needed!
?. [SP3 Protocol Day 1]
After the final rinse, be sure to remove as much of the Ethanol supernatant as possible without disturbing the beads.
You should not leave more than   in the tube.
?. [SP3 Protocol Day 1]
Incubate the samples in the Thermoshaker for 10 min  OR sonicate your samples for  at  .
Centrifuge: 750 33, 37 10
37 Room temperature
This is to disaggregate the beads and make them more amenable to pipetting.
?. [SP3 Protocol Day 1]
Remove the trypsin solution from the freezer for thawing.
Trypsin should be removed just prior to use (leaving enough time to thaw) and then immediately returned to the freezer.
?. [SP3 Protocol Day 1]
Add  of the  trypsin solution to each tube.
1 µl
This will end up with  of trypsin to each sample. To use a different amount, adjust the amount of trypsin accordingly.
?. [SP3 Protocol Day 1]
Incubate your samples at the Thermoshaker at .
Centrifuge: 750 33, 1080 min, 37 10
Reminder: The samples cannot be stored or frozen until the beads have been removed.
?. [SP3 Protocol Day 2]
General Information and preperation:All preparations and pretreatment steps should take place in a dedicated extraction hood.Wipe down all surfaces (1. 5% (v/v) bleach, 2. ultrapure water, 3. 70% (v/v) Ethanol).Alliquot out the reagents that you need (5% (v/v) TFA, Methanol, 60% (v/v) ACN 0,1% (v/v) TFA, 3% (v/v) ACN 0,1% (v/v) TFA).Check if you have prepared StageTips, clean magnetic racks, and clean StageTip holders.
?. [SP3 Protocol Day 1]
Mix your sample again through up and down pipetting to ensure an equal distribution of the beads and the trypsin.
?. [SP3 Protocol Day 2]
Add of ACN,  TFA to the tips and centrifuge.
150 µl
Centrifuge: 2000 34, 1 min
Check your StageTips after centrifugation to see if all the liquid went through and that the StageTip is still intact.