TITLE: Ancient Proteins Extraction Protocol
AUTHORS: Ashley Scott, Christina Warinner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for extracting total proteins from archaeological dental calculus. It is based on the filter-aided sample preparation (FASP) protocol first published by Wisniewski et al. 2009. Specific modifications have been made to enable protein extraction from calcified material and to ensure recovery of ancient proteins. </div></div>

[STEPS]
?. [Sample Prep]
Weigh samples (aim for 5-10 mg per sample) and place within a 1.5 mL Safelock Eppendorf tube.
?. [Sample Prep]
Also prepare empty tubes for blank extractions that should be processed alongside your samples to monitor for lab contamination
?. [Demineralization]
Add 500 µL of 0.5 M EDTA to each sample, including extraction blanks.
?. [Demineralization]
Close tubes tightly and set on a rotator until calculus becomes completely dissolved (invisible), or buoyant and feathery, typically between 2-5 days.
?. [EXTRACTION DAY 1]
Prepare solutions and label all tubes that will be needed for Day 1
?. [EXTRACTION DAY 1]
Spin down decalcified samples at 14,000 rcf for 5 minutes
?. [EXTRACTION DAY 1]
Remove 300uL of EDTA supernatant and place in a new labeled sample and store in -20˚C freezer as backup.
?. [EXTRACTION DAY 1]
Add 50 µL of UA solution to a Microcon 30kDa filter unit. Avoiding the pellet, transfer the remaining 200 uL EDTA supernatant to the UA in the filter unit. Resuspend thoroughly to mix.
?. [EXTRACTION DAY 1]
Spin at 14,000 rcf at 18˚C for 15-18 minutes.
?. [Extraction Day 1: Lysis and Denaturation]
To the remaining pellet, add 30 µL of SDS-lysis buffer and mix by resuspension. Incubate on heat block for 5 minutes at 95°C.
?. [Extraction Day 1: Lysis and Denaturation]
Centrifuge at 14,000 rcf at 18˚C for 10 minutes.
?. [Extraction Day 1: Lysis and Denaturation]
Add 200 µL of UA solution to the filter unit used in Step 7, followed by the pellet supernatant (~30 µL), avoiding any pellet debris, and resuspend to mix.
?. [Extraction Day 1: Lysis and Denaturation]
Centrifuge filter unit at 14,000 rcf at 18˚C for 15-18 min until all liquid has passed through
?. [Extraction Day 1: Lysis and Denaturation]
Discard flow-through
?. [Extraction Day 1: Lysis and Denaturation]
Add another 200 µL of UA solution to the filter unit
?. [Extraction Day 1: Lysis and Denaturation]
Centrifuge filter unit at 14,000 rcf at 18˚C for 15-18 min until all liquid has passed through
?. [Extraction Day 1: Lysis and Denaturation]
Place the remaining pellet tube in -20°C freezer for storage.
?. [Extraction Day 1: Alkylation]
Add 100 µL IAA solution (0.05 M) to the filter unit.
?. [Extraction Day 1: Alkylation]
Mix at 300 rpm in the thermo-mixer for 1 min in the dark (cover with foil).
?. [Extraction Day 1: Alkylation]
Incubate for 15-20 min in the dark
?. [Extraction Day 1: Alkylation]
Centrifuge at 14,000 rcf at 18˚C for 12-15 min until all liquid has passed through.
?. [Extraction Day 1: Alkylation]
Discard flow-through in halogenated waste
?. [Extraction Day 1: Wash steps]
Add 200 µL of urea solution to the Microcon unit and centrifuge at 14,000 rcf for 15-18 minutes.
?. [Extraction Day 1: Wash steps]
Repeat for a total of three washes of urea solution. Discard flow-through.
?. [Extraction Day 1: Wash steps]
Add 100 µL 0.05M TEAB to the Microcon unit and centrifuge at 14,000 rcf for 12-15 minutes.
?. [Extraction Day 1: Wash steps]
Repeat twice for a total of three washes of TEAB solution.
?. [Extraction Day 1: Digestion]
Add 100uL of trypsin solution to each filter unit
?. [Extraction Day 1: Digestion]
Incubate overnight at 37C in the thermo-mixer at 300 rpm
?. [Extraction Day 2]
Transfer the Spin Filter to a new labeled collection tube.
?. [Extraction Day 2]
Add 40 µL of TEAB Solution. Centrifuge the Spin Filter at 14,000 rcf for 10 min. DO NOT DISCARD FLOW-THROUGH.
?. [Extraction Day 2]
Repeat this step once.
?. [Extraction Day 2]
Add 50 μL 0.5 M Sodium Chloride Solution and centrifuge the Spin Filter at 14,000 rcf for 10 min.
?. [Extraction Day 2]
The filtrate contains digested proteins. Acidify the filtrate with TFA to a pH below 3 (approximately 18 μL of TFA will be needed).
?. [Extraction Day 2]
Desalt using method of choice (e.g., StageTips or ZipTips).