Jun 30, 2025
Suspension Trapping for Archaeological Bone Proteins
- Ian Engels1,
- Alexandra Burnett1,
- Rachel Siân Dennis1
- 1Ghent University
Protocol Citation: Ian Engels, Alexandra Burnett, Rachel Siân Dennis 2025. Suspension Trapping for Archaeological Bone Proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge8qq7g47/v1
Manuscript citation:
Engels, I., Burnett, A., Robert, P., Pironneau, C., Abrams, G., Bouwmeester, R., Van der Plaetsen, P., Di Modica, K., Otte, M., Straus, L.G., Fischer, V., Bray, F., Mesuere, B., De Groote, I., Deforce, D., Daled, S., and Dhaenens, M. (2025). Classification of Collagens via Peptide Ambiguation, in a Paleoproteomic LC-MS/MS-Based Taxonomic Pipeline. Journal of Proteome Research, 24(4), pp.1907-1925. https://doi.org/10.1021/acs.jproteome.4c00962
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We routinely use this protocol and it's working.
Created: June 27, 2025
Last Modified: June 30, 2025
Protocol Integer ID: 221178
Keywords: Palaeoproteomics, Proteomics, Archaeology, Bone, suspension trapping for archaeological bone protein, archaeological bone protein, archaeological bone, abundant collagen peptides for species identification, abundant collagen peptide, dentine sample, dentine samples for analysis, protein, suspension trapping, peptide
Funders Acknowledgements:
Special Research Fund (BOF), Ghent University
Grant ID: BOF.GOA.2022.0002.03
Abstract
'Suspension Trapping for Archaeological Bone Proteins' is a suspension trapping-based protocol developed to prepare archaeological bone and dentine samples for analysis by LC-MS/MS. This straightforward protocol features a rapid digestion time and does not use any chemicals that are highly toxic. The authors use this protocol routinely to extract abundant collagen peptides for species identification by ZooMS/MS.
Guidelines
This protocol is designed to be used for powdered bone samples of 5-20 milligrams. You may wish to increase the reagent volumes if your sample is significantly larger.
Materials
Invitrogen™ UltraPure™ SDS Solution, 10%InvitrogenCatalog #15-553-027 Hydrochloric acid 1.2 mol/lChem-Lab Analytical bvbaCatalog #CL05.0312.1000
AcetoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #179124
Triethylammonium bicarbonate bufferMerck MilliporeSigma (Sigma-Aldrich)Catalog #90360-100ML
DTT for Biochemistry 99+% C4H10O2SChem-Lab Analytical bvbaCatalog #CL00.0481
S-Methyl methanethiosulfonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #64306-10ML
Phosphoric acid 85% a.r.Chem-Lab Analytical bvbaCatalog #CL00.0605.1000
Methanol, LC-MS gradeChem-Lab Analytical bvbaCatalog #CL00.1377.1000
Trypsin/Lys-C Mix, Mass Spec Grade, 5 x 20ugPromegaCatalog #V5073
Formic acid 0.1% in Water HPLCBiosolveCatalog #232406
Acetonitrile, LC-MS gradeChem-Lab Analytical bvbaCatalog #CL00.0194.1000
Equipment
1.5mL Epps - Eppendorf Protein LoBind Tube (Safe-Lock, PCR clean)
Suspension trapping columns - Magen Biotechnology Co. Ltd. (China) HiPure Viral Mini Columns with 2mL collection tubes; Cat No. C13112
MS vials - Verex Vial, 9mm Screw, PP, 300uL, Clear, No Patch, 1000/pk; Part: AR0-39P2-13.
Centrifuge - Eppendorf Centrifuge 5417R
CentriVap - Thermo Scientific Savant SPD111V SpeedVac Concentrator
Thermomixer - Eppendorf Thermomixer Comfort
Vortexes - Scientific Industries Vortex-Genie 2
Sonicator - Elma Transsonic 460
Equipment
Verex Vial, 9mm Screw, PP, 300uL, Clear, No Patch, 1000/pk
NAME
Vial
TYPE
Verex
BRAND
AR0-39P2-13
SKU
LINK
Equipment
HiPure Viral Mini Column
NAME
Trapping Column
TYPE
Magen
BRAND
C13112
SKU
LINK
Troubleshooting
Safety warnings
Wear appropriate protective gear when performing this protocol, minimally a lab coat and gloves, and preferably goggles, closed shoes, and long leg coverings.
Before start
We advise you to prepare all reagents except the MMTS before starting your protocol. The lysis buffer in particular can take a long time to come into solution when preparing from solid SDS.
Lysis Buffer - 10% SDS in 100mM TEAB
Binding/Washing Buffer - 100mM TEAB in 90% MeOH
Demineralisation
Add 600µL of 0.6M HCl to each 1.5mL Protein Lo-Bind Eppendorf containing 5mg of bone powder. Vortex to suspend the powder in the acid.
Note
Bone powder can be highly static. To avoid sample loss, pipette the acid onto the side of the Eppendorf above the fluid.
Incubate overnight on a thermomixer at 650rpm.
Centrifuge at 15000g for 10 minutes
Pipette the supernatant off. You may discard this or separate into clean Eppendorfs for later analysis.
Wash the pellet by adding 500µL of ice-cold acetone, then centrifuge for 10 minutes at 15000g, and pipette the acetone off into a waste beaker. Leave the tubes open to dry out the pellet.
Solubilise, Reduce, Alkylate
Add 25.5µL lysis buffer solution (5% SDS + 50mM TEAB).
Resuspend the pellet by vortexing and sonicating the sample thoroughly.
Add DTT to a final concentration of 5mM.
Incubate in a thermo-mixer for 30 minutes at 37°C, at 600rpm.
Add MMTS to a final concentration of 10mM.
Incubate for 10 minutes in darkness while mixing at 600rpm.
Protein washing
Acidify the samples by adding 27.5% phosphoric acid until the pH is 1 or lower.
Add 165µL binding/washing buffer (100mM TEAB in 90% methanol) to the sample and gently vortex to mix.
Note
You may notice floculation of your sample at this point. This is normal. All of the fluid and floculated material should be transferred in the next step.
Transfer the sample to the suspension trapping column.
Note
Be very careful not to touch or disrupt the column filter with your pipette tip at any point. If this is significantly damaged the filter may perform poorly, resulting in peptide loss.
Centrifuge for 30 seconds at 4000g.
Add 150µL of washing/binding buffer to the column and centrifuge again for 30 seconds at 4000g.
Repeat the previous step once or twice (depending on the sample). You may need to empty the waste reservoir.
Dry the filter by centrifuging for 1 minute at 4000g.
Digestion
Transfer the trapping columns to fresh 1.5mL Protein lo-bind Eppendorfs.
Prepare your digestion solution: suspend your lyophilised Trypsin/Lys-C in 50mM TEAB at a concentration of 0.0125µg/µL [i.e. for a vial of 20µg protease, add 1.2mL TEAB].
Add 40µL of digestion solution (equal to 0.5µg Trypsin/Lys-C) to the column filters.
Note
You may modify the quantity of trypsin in your samples but the quantity of digestion solution added should be 40µL.
Incubate for 2 hours at 47°C.
Elution
Add 30µL of 50mM TEAB, incubate for 1 minute, then centrifuge for 1 minute at 4000g.
Add 30µL of 0.1% FA, incubate for 1 minute, then centrifuge for 1 minute at 4000g.
Add 30µL of 50% ACN, incubate for 1 minute, then centrifuge for 1 minute at 4000g.
Dry the sample in a SpeedVac. The dried sample may be stored at -20°C (or colder) until resuspension.
Resuspension
Add 20µL of 0.1% FA.
Vortex and sonicate the sample thoroughly to resuspend the peptides.
Centrifuge for 15 minutes at 15000g.
Transfer the sample to MS vials ready for analysis.
Protocol references
This protocol was developed by Ian Engels, drawing on existing protocols by Simon Daled & Alexandra Burnett (see reference), and Rachel Siân Dennis. RSD gave advice on the implementation of the suspension trapping portion of the protocol and AB contributed to minor protocol revisions.
Daled, S. & Burnett, A., (2023). Protein Extraction from Dental Enamel [protocol], 21 Aug 2023. protocols.io. dx.doi.org/10.17504/protocols.io.8epv5j8n4l1b/v2
This protocol is written as employed in the following publication:
Engels, I., Burnett, A., Robert, P., Pironneau, C., Abrams, G., Bouwmeester, R., Van der Plaetsen, P., Di Modica, K., Otte, M., Straus, L.G. and Fischer, V., (2025). Classification of Collagens via Peptide Ambiguation, in a Paleoproteomic LC-MS/MS-Based Taxonomic Pipeline. Journal of Proteome Research, 24(4), pp.1907-1925. https://doi.org/10.1021/acs.jproteome.4c00962