TITLE: Protein Extraction from Dental Enamel
AUTHORS: simon.daled, Alexandra Burnett

[DESCRIPTION]
This protocol details a method for extracting proteins from ~5mg of powdered dental enamel for proteomic analysis by LC-MS/MS. This procedure includes the use of alkylating and reducing agents, a sample cleanup step, and features the optional addition of synthetic peptide heavy standards for absolute quantification and quality control of the analysis. 

Total protocol duration is dependent on the number of samples to be processed. Protein extraction will typically take a day-and-a-half, after which time the samples can be safely stored prior to instrumental analysis. 

[GUIDELINES]
Always process blank samples alongside your enamel samples to allow for the observation of laboratory contamination.

[STEPS]
SECTION: Sample acquisition
1. 
 
Place your tooth on a fresh piece of weighing paper on a clean worktop. 
Using a small drill bit, gently abrade the surface of the tooth crown to remove powdered enamel and collect it on the weighing paper.  
SECTION: Sample acquisition
2. Carefully tip the powdered enamel into a protein lo-bind Eppendorf tube. Weigh the tube and record the weight of the powdered enamel inside.
SECTION: Protein Extraction
4. Demineralisation
Add 200µL of 1.2M HCl to each sample tube. Vortex the tubes briefly to mix.
Incubate for two hours in a thermo-mixer, using moderate agitation at room temperature.
SECTION: Protein Extraction
3. 
 

 

SECTION: Protein Extraction
5. Precipitation
Add 100% TCA (in water) to a final concentration of 33% (V/V) (i.e. 100µL).
Incubate on ice for one hour.
Centrifuge for 10 minutes at 15,000G at 0°C.
Pipette off the supernatants and discard (or retain and freeze if desired).

SECTION: Protein Extraction
6. Wash the pellet
Pipette 500µL of ice-cold acetone into each sample tube.
Centrifuge for 10 minutes at 15,000G.
Pipette off the supernatants and discard (or retain if desired).
SECTION: Protein Extraction
7. Resuspend in buffer
Add 200µl of 8M urea in 1M ABC buffer (pH 8).
Vortex and sonicate until all pellets are resuspended.
SECTION: Protein Extraction
8. Reduction
Add DTT to a final concentration of 5mM.
Incubate for 30 minutes at 37°C.
SECTION: Protein Extraction
9. Alkylation
Create a fresh stock solution of MMTS in 2-propanol (200mM).
Add MMTS to a final concentration of 10mM in each sample.
Incubate at room temperature in the dark for 15 minutes. 
 

SECTION: Protein Extraction
11. Digestion
Prepare a solution of trypsin, or trypsin and lysine-C, in 1M ammonium bicarbonate.
Add enzyme to a 1:25 (w/w) enzyme/protein ratio (around 1-2µg per sample).
Incubate overnight at 37°C. If there is visible debris in the tubes following digestion, sonicate until this is broken up.
SECTION: Protein Extraction
13. Drying down
Vacuum-dry the samples. Drying at up to 40°C can accelerate the process without damaging the peptides.
 

SECTION: Resuspension for LC-MS/MS
14. [Optional] Prepare a solution containing amelogenin heavy labelled peptide standards in 0.1% formic acid. Add heavy peptides to a concentration of around 5fmol/µL, depending on the LOD and sensitivity of the LC-MS/MS system. 

Add between 10-30µL of 0.1% formic acid [and heavy standards] to each sample tube.
 

Sonicate and vortex the samples to ensure thorough solubilization.
Centrifuge at 16,000G for 10 minutes.
SECTION: Resuspension for LC-MS/MS
15. Transfer the samples to MS sampling vials ready for analysis.
 

SECTION: Protein Extraction
12. Sample Cleanup via Solid-Phase Extraction [Optional]
Condition one Strata tube per sample with 1mL MeOH.
Equilibrate the Strata tubes twice with 1mL ultrapure water.

 
Remove the waste MeOH and water and replace the waste collection tubes with fresh Eppendorf lo-bind tubes.
Slowly load 1mL of sample per tube.
Wash twice with 1 mL of 5% MeOH in ultrapure water. [The cartridge can be allowed to run dry at this stage.]
Elute the sample with 1 mL of 1% FA in MeOH; first let a small amount soak the cartridge for 2 minutes and then slowly elute the sample with the rest of the elution buffer.
SECTION: Protein Extraction
10. Buffer dilution
Dilute the samples to a final concentration of 2M urea with 1M ABC buffer.