TITLE: Zooarchaeology by Mass Spectrometry (ZooMS) for bone material - Acid soluble protocol
AUTHORS: Samantha Brown, Sandra Hebestreit, Naihui Wang, Nicole Boivin, Katerina Douka, Kristine Richter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection details the different established protocols for Zooarchaeology by Mass Spectrometry (ZooMS) for use on archaeological bone.  ZooMS allows for taxonomic identification by the peptide mass fingerprinting of collagen type I.  These protocols can be used individually or combined depending on the preservation, sample size, and ability to do destructive analysis.   All the protocols are optimized for bone as the starting material.</div><div class = "text-block">In the acid soluble protocol bone is pretreated with hydrocholoric acid.  The acid is removed and filtered leaving the collagen which is then digested with trypsin.  The peptides are purified using C18 ZipTips.  This protocol can be used in conjuntion with the acid insoluble protocol which analyzes the bone shaddow left after the acid is removed.</div><div class = "text-block">This protocol is suitable for a wide range of preservation conditions from very good to poor preservation.  It works in cases where the bone is too fragile or the preservation is too poor for a collagen shaddow to remain after demineralization.  The ideal starting volume is 10-20mg of bone as a powerder or a bone chip, but in cases of very poor preservation, a larger starting volume may be required.</div><div class = "text-block">If you are using this protocol, please cite the DOI for the protocol and the following two papers on which it is based:</div><div class = "text-block"><span>Buckley, M., Collins, M., Thomas-Oates, J., & Wilson, J. C. (2009). Species identification by analysis of bone collagen using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. </span><span style = "font-style:italic;">Rapid Communications in Mass Spectrometry: RCM</span><span>, </span><span style = "font-style:italic;">23</span><span>(23), 3843–3854. https://doi.org/10.1002/rcm.4316</span></div><div class = "text-block"><span>van der Sluis, L. G., Hollund, H. I., Buckley, M., De Louw, P. G. B., Rijsdijk, K. F., & Kars, H. (2014). Combining histology, stable isotope analysis and ZooMS collagen fingerprinting to investigate the taphonomic history and dietary behaviour of extinct giant tortoises from the Mare aux Songes deposit on Mauritius. </span><span style = "font-style:italic;">Palaeogeography, Palaeoclimatology, Palaeoecology</span><span>, </span><span style = "font-style:italic;">416</span><span>, 80–91. https://www.sciencedirect.com/science/article/pii/S0031018214003071</span></div></div>

[STEPS]
?. [Reagent preparation]
General Information:We recommend to use glassware for buffers and solutions and no long-term storage in plastic containers and/or tubes.TFA, HCl and ACN stock solutions should be handled under a fume hood only.Always label all chemicals with the compound name, concentraion and the date.Chemicals are prepared with water from an ultrapure water system (e.g. Milli-Q water, see materials list)
?. [Reagent preparation]
Hydrochloric acid (HCl) (Shelf life: 6 months at )
4 °C
Handling of HCl should be performed under a fume hood because of its corrosive properties. HCl can cause severe skin burns, eye and respiratory irritations. Don´t touch your skin or eyes with contaminated gloves and change them immediately.
?. [Reagent preparation]
For 1000 ml of 0.6 M HCl, add  of  HCl to  ultrapure water carefully. Mix gently.
50 mL
950 mL
Please always calculate the needed amount of HCl stock solution you need since this is dependent of the specific compound density.
?. [Reagent preparation]
Ammonium Bicarbonate (AmBiC) buffer,  (Shelf life: 6 months at )
4 °C
?. [Reagent preparation]
For 1000 ml, weigh out  of AmBiC powder and transfer it into a clean glass bottle.
3.95 g
?. [Reagent preparation]
Dissolve the AmBiC in of ultrapure water and mix by genty inverting.
800 mL
?. [Reagent preparation]
Fill up to  with ultrapure water and mix by gently inverting.
1000 mL
?. [Reagent preparation]
Trifluoroacetic acid (TFA) (Shelf life: 6 months at  )
0 Room temperature
Triflouroacetic acid (TFA) is very hazardous (corrosive)! Handling should only be performed under a fume hood.Prepare this solution in a brown glass bottle since TFA is slightly 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.
?. [Reagent preparation]
Dilute  TFA stock solution using a glass syringe/pipette (no plastic) in   ultrapure water and mix carefully.
1 mL
19 mL
?. [Reagent preparation]
Wash solution:  TFA in ultrapure water (Shelf life: 6 months at   )
0 Room temperature
?. [Reagent preparation]
For 1000 ml, add  of TFA to  of ultrapure water and mix by gently inverting.
1 mL
1000 mL
?. [Reagent preparation]
Conditioning solution:  TFA in  Acetonitrile (ACN), (Shelf life: 3 month at  )
0 Room temperature
ACN is a volatile compound and if there is too much headspace in the storage bottle the concentration of ACN in solution goes down over long-term stoorage. We recommend not to store small solution amounts in large bottles over a long period of time. Either prepare new conditioning solution or store the remaining solution in smaller bottles.
?. [Reagent preparation]
For 1000 ml, mix  ACN with  ultrapure water.
500 mL
500 mL
?. [Reagent preparation]
Add  of TFA carefully and mix by genty inverting.
1 mL
?. [Reagent preparation]
Trypsin solution (Stable once resuspended for 1 month at  )
-20 °C
?. [Reagent preparation]
Freeze dried Trypsin () is resuspended in   of Trypsin resuspension buffer (included in Trypsin Kit or per manufactures instructions). The final concentration is now  .
20 µg
50 µl
?. [Reagent preparation]
Transfer the solution in a labelled 0.5 ml tube and store at  .
-20 °C
?. [Sample preparation and collagen extraction]
Weighing out the sample
General informationAll steps should take place in a dedicated wet chemistry laboratory.Wipe down all surfaces with 1. ultrapure water, 2. 70% (v/v) ethanol).Alliquot out the reagents you will need (HCl).Needed after proper bone material deminaralization (see step 13): Aliquot out the reagents you will need (AmBic).
?. [Sample preparation and collagen extraction]
Archaeological samples: Weigh out  -  of bone chips or ~  bone powder into a microcentrifuge tube labelled with the sample name, date and your name. Record the weight.Modern samples: Weigh out - of bone chips or ~ bone powder into a microcentrifuge tube labelled with the sample name, date and your name. Record the weight.
10 mg
20 mg
10 mg
5 mg
15 mg
7 mg
?. [Sample preparation and collagen extraction]
Acid Demineralization (destructive)Add  of cold ()  HCl to the sample (or enough acid to cover the sample completely) and place the tubes in the fridge at . Leave until demineralization is complete (bone becomes flexible/ spongy and no bubbling is visible anymore).
500 µl
4 °C
4 °C
This may take 1 day to 2 weeks for specific sample types. Less time will be needed for bone powders (4-10 h).
For samples with good collagen preservation, complete demineralization is not necessary. Please be aware that the more the collagen remains in acid the more it breaks down. Hence monitor samples carefully at this stage to reduce protein loss.
Don´t forget to prepare blanks at this point! This should be empty tubes with only HCl added. Perform every step with the blanks, which you do with the actual samples.
?. [Sample preparation and collagen extraction]
Centrifuge the sample for  to settle the bone and (heavier) collagen to the base of the tube.
Centrifuge: 20000 33, 1 min
?. [Sample preparation and collagen extraction]
Remove the acid supernatant into a new tube. We recommend to label this tube with the sample number and the suffix "AC",
Do not disturb the bone residues from your sample with the pipette.
If you want to analyse the acid insolube collagen fraction (please see the protocol "Zooarchaeology by Mass Spectrometry- Acid insoluble protocol") you should use this bone chip/bone powder fraction and store it at   .
?. [Sample preparation and collagen extraction]
Buffer exchangePlace 30 kDa ultrafilters into labelled tubes.
?. [Sample preparation and collagen extraction]
Centrifuge at  until acid has been pushed through the filter. This can take between  and .
Centrifuge: 3700 33
Put on cap and place filters into the centrifuge with the cap strap toward the center of rotor, this ensures the special shaped ultrafilters will fit properly into the centrifuge.
You should not let the filter dry out during this stage. Small amounts of liquid above the filter are fine.
?. [Sample preparation and collagen extraction]
Discard the flow-through.
?. [Digestion and peptide clean up]
Trypsin DigestionAdd  of  trypsin solution to the “EXT” labelled tube.
1 µl
?. [Digestion and peptide clean up]
Incubate sample overnight at  (approx. -).
37 °C
?. [Digestion and peptide clean up]
Centrifuge your samples  after incubation.
Centrifuge: 20000 33, 1 min
?. [Digestion and peptide clean up]
Aliquot out the TFA you need and add  of  TFA to stop the trypsin.
1 µl
The peptides can be purified using a C18 ZipTip. If not extracting immeditely, the samples can be stored in the freezer at   at this point.
?. [Digestion and peptide clean up]
Preperations for C18 ZipTippingFor each sample you must prepare separate tubes of wash and conditioning solution. Prepare as follows:One tube per sample containing  conditioning solution (or you can just use a common reagent reservoir - label with "C")One tube per sample containing  wash solution (label with "W")One tube per sample  with  conditioning solution- this tube is to collect your purified peptides. We recommend to label it with the prefix “COL”  and the sample name.
250 µl
450 µl
50 µl
General information:During your extraction your peptides are bound to the C18 filter sitting in the pipette tip (ZipTip).The peptides then need to be washed and eluted. Therefore, subsequent wash and conditioning solutions will be ‘contaminated’ with your sample and so solutions after the sample has been bound to the C18 filter and cannot be used for more than one sample.
?. [Digestion and peptide clean up]
C18-ZipTipping Purify your peptides using the C18 ZipTip from the digested “EXT” sample as follows:Rinse tip twice with  of conditioning solution and discard.Rinse tip twice with  of wash solution and discard.Rinse tip twice with  of wash solution and discard.Elute sample into the prepared ‟COL” labelled tube with the prepared  of conditioning solution and pass through tip at least 5 times.
Before use, each tip needs to be conditioned. A separate tip is used for each blank and sample. The pipette should be set at   and the volume NOT changed during the time. Be careful to avoid pulling air through the filter, especially at the steps with only   of solution. We recommend to use an extra ZipTipping Only pipette as repeated use of the filters pulls the pipettes out of calibration.
100 µl
100 µl
100 µl
50 µl
Be careful not to push air through the C18 filter (never go past the first stop point of the pipette)!
The sample(s) are now ready to be spotted on the MALDI plate. If not spotting immediately, store sample(s) in freezer at  .
?. [Sample preparation and collagen extraction]
Add  of  AmBic and centrifuge at until AmBic has passed through filter. This can take between  and .
500 µl
Centrifuge: 3700 33
?. [Sample preparation and collagen extraction]
Discard the flow-through.
?. [Sample preparation and collagen extraction]
Add  of  AmBic to the filter and mix carefully by pipetting.
100 µl
If your sample amount is limited or if less sample then recommended was available, use 50 µl  of AmBic.
?. [Sample preparation and collagen extraction]
Label two new sets of microcentrifuge tubes in the meantime.
These tubes are to cellect the AmBic supernatant from your filter (see step 12.8). We recommend to label one set with the suffix “EXT” (extract) and the sample name and the second set with the suffix “SE” (second extract) and the sample name. If you are going to use 50 µl of AmBic for eluting the collagen from the filter (see step 12.7), only the EXT tube is needed.
?. [Sample preparation and collagen extraction]
Transfer now  of the AmBic supernatant to the “EXT” labelled tube and the remaining  supernatant to the  “SE” labelled tube.
50 µl
50 µl
The “SE” labelled tube serves as back up and can be stored at  . Adjust the amounts of "EXT" and "SE" if less AmBic was used ( see step 12.6 and see the note).
?. [Sample preparation and collagen extraction]
Take  of acid supernatant and put into a 30 kDA ultrafilter.
250 µl
Take care not to touch the tip to the membrane.
For well-preserved collagen samples a 30 kDA ultrafilter is sufficient. Smaller sized filters easily get clogged. For poorly preserved collagen, a 3 kDA or 10 kDA ultrafilter can be considered.