Mar 03, 2025
Shotgun proteomics for archaeological bone material - in solution protocol for bone samples from tropical sites
- 1Australian National University
Protocol Citation: Sofia Samper Carro 2025. Shotgun proteomics for archaeological bone material - in solution protocol for bone samples from tropical sites. protocols.io https://dx.doi.org/10.17504/protocols.io.81wgbrjn1lpk/v1
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 use this protocol and it's working
Created: February 27, 2025
Last Modified: March 03, 2025
Protocol Integer ID: 123481
Keywords: zooarchaeology, palaeoproteomics, LC MS/MS, tropical
Funders Acknowledgements:
Australian Research Council
Grant ID: CE170100015
Abstract
The analysis of bone ancient proteins (palaeoproteomics) is revolutionising the design of research strategies for zooarchaeological assemblages worldwide. By providing means to identify species presence in sites from samples that are not identifiable macroscopically through traditional methods, palaeoproteomics is offering a new exciting approach to understand people’s past interactions with their environments.
Nevertheless, this technique is not depleted of challenges. Proteins preserved in ancient bones have undergone several modifications, most of which we do not know yet how they change the organic bone component. As a relatively new technique, much progress is needed in the optimization and development of
laboratory protocols tailored to different taphonomic histories.
With a focus in European context, they are currently just few examples of the application of this technique in non-temperate contexts.
This protocol presents a modified version of a common pipeline to prepare bone samples documented in tropical archaeological or palaeontological sites for LC MS/MS analysis. The main differences with other laboratory protocols consist of reducing washings steps, avoiding reduction/alkylation processes, optimizing protein digestion and concentrating eluted samples.
This protocol has been successfully tested at the Australian National University, obtaining positive results for archaeological bone samples from Northwest Australia, Timor-Leste, PNG and Indonesia.
Image Attribution
Image created by Sofia Samper Carro with a free Biorender licence
Guidelines
1. Perform all steps in a standard wet chemistry laboratory, as a minimum biosafety level
2. Wear PPE while conducting all the steps (laboratory coat, gloves, hairnet and safety googles)
3. Be aware of any specific regulation in the handling, storage and disposal of reagents and chemical waste
Materials
- Standard glass bottles in different sizes (500 ml, 250 ml, 100 ml, 50 ml, 20 ml)
- Glass screw top microvials with insert for <2ml samples, e.g. SureSTART 0.3ml Thermo Scientific
- 9mm Screw caps with septum, e.g. SureSTART 9mm Screw Caps Thermo Scientific
- Pipette tips for different voluminal (0.5 µl - 5 ml) e.g. from Corning Deckworks
- Pipettors with different voluminal ranges (0.5- 10 µl, 2-20 µl, 20-200 µl, 100-1000 µl, 0.5-5 ml), e.g. Eppendorf Research‱ plus
- LoBind Microcentrifuge tubes e.g 1.5 ml and/or 2.0 ml, safe lock, Eppendorf
- Standard tube racks for microcentrifuge
- pH strips, e.g. VMR (ranges from 0-14)
- Scale for laboratory use, e.g. Metler Toledo precision scale
- Centrifuge with a rotor for 1.5 ml/2.0 microcentrifuge tubes, e.g. Eppendorf 5425 with rotor FA-24x2
- Reagent reservoirs, e.g. disposable reagent reservoirs, VWR
- Ultrapure water system, e.g. Elga Purelab Quest, Type 1 water system with UV lamp
- Vaccum centrifuge, e.g. Labconco Centrivap Acid Resistant Centrifugal Vacuum Concentrator
- Incubator, e.g. Eppendorf Thermomixer C
- Clean Spot PCR workstation 240v
- Agate mortar and pestle
- Ultrasonic bath, eg. Vevor 30L
Protocol materials
Pierce Trypsin ProteaseThermo Fisher ScientificCatalog #90057
Step 15
Glacial acetic acidFisher ScientificCatalog #A38-500
Step 16
Trifluoroacetic acid (TFA)Bio Basic Inc.Catalog #TC8960.SIZE.100mL
Step 22
Formic acid, LC-MS gradeThermo Fisher ScientificCatalog #28905
Step 27
Acetonitrile (ACN)Fischer ScientificCatalog #10660131
Step 27
Ammonium bicarbonate (NH4HCO3)Merck MilliporeSigma (Sigma-Aldrich)Catalog #AC393212500
Step 8
10% Bleach
Step 1.1
70% isopropyl alcohol
Before starting
70% Ethanol
Before starting
Hydrochloric acidMerck MilliporeSigma (Sigma-Aldrich)
Step 5
Safety warnings
Be aware of your country and facility specific chemical safety guidelines.
This protocol uses several solvents, acids and other chemicals which need special precaution. Please be aware of the international GHS hazard statements (listed below) and follow your country and institute specific precautions/guidelines.
The GHS hazard (H-) and precautionary (P-) statements for the chemicals used in this protocol, are:
Ethanol (for cleaning):
- H225 Highly Flammable liquid and vapor
- H319 Causes serious eye irritation
- P210 Keep away from heat, hot surface, sparks, open flames and other ignition sources. - No smoking)
- P264 Wash ... thoroughly after handling
- P280 Wear protective gloves/protective clothing/eye protection/face protection
- P303+P361+P353 IF ON SKIN (or hair): Take off Immediately all contaminated clothing. Rinse SKIN with water or shower
- P337+P313 IF eye irritation persists: Get medical advice/attention
Isopropyl alcohol (for cleaning):
- H225 Highly Flammable liquid and vapour
- H319 Causes serious eye irritation
- H336 May cause drowsiness or dizziness
- P210 Keep away from heat/sparks/open flames/hot surfaces. — No smoking.
- P261 Avoid breathing dust/fume/gas/mist/vapours/spray.
- P280 Wear protective gloves/protective clothing/eye protection/face protection.
- P303+P361+P353 IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower.
- P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
- P337+P313 IF eye irritation persists: Get medical advice/attention.
- P370+P378 In case of fire: Use … for extinction.
- P403+P235 Store in a well-ventilated place. Keep cool.
- P405 Store locked up.
Bleach (for bone decontamination)
- H314 Causes severe skin burns and eye damage.
- H400 Very toxic to aquatic life
- P260 Do not breathe vapour/ spray.
- P264 Wash contaminated skin thoroughly after handling.
- P273 Avoid release to the environment.
- P280 Wear protective gloves/ protective clothing/ eye protection/ face protection.
- P301+P330+P331 IF SWALLOWED: Rinse mouth. Do NOT induce vomiting.
- P303+P361+P353 IF ON SKIN (or hair): Take off immediately all contaminated clothing. Rinse skin with water/ shower.
- P304+P340 IF INHALED: Remove victim to fresh air and keep at rest in a position comfortable for breathing.
- P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing.
- P310 Immediately call a POISON CENTER or doctor/ physician.
- P321 Specific treatment
- P363 Wash contaminated clothing before reuse.
- P391 Collect spillage.
- P405 Store locked up.
- P501 Dispose of contents/ container in accordance with national regulations.
Hydrochloric acid:
- H290 May be corrosive to metals
- H314 Causes severe skin burns and eye damage
- H335 May cause respiratory irritation
- P280 Wear protective gloves/protective clothing/eye protection/face protection
- P301+P330+P331 IF SWALLOWED: Rinse mouth. Do NOT induce vomiting
- P305+ P351+ P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do - continue rinsing
- P308+P310 IF exposed or concerned: Immediately call a POISON CENTER or doctor/physician
Ammonium bicarbonate (AmBic):
- H302 Harmful if swallowed
- P264 Wash thoroughly after handling
- P270 Do not eat, drink or smoke when using this product
- P301+P312 IF SWALLOWED: call a POISON CENTER/doctor/... IF you feel unwell
- P330 Rinse mouth
Acetonitrile:
- H225 Highly Flammable liquid and vapor
- H302+H312+H332 Harmful if swallowed, in contact with skin or if inhaled
- H319 Causes serious eye irritation
- P210 Keep away from heat, hot surface, sparks, open flames and other ignition sources. - No smoking
- P280 Wear protective gloves/protective clothing/eye protection/face protection
- P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do - continue rinsing
- P403+P235 Store in a well-ventilated place. Keep cool.
Trifluoracetic acid:
- H318 Causes serious eye damage
- H314 Causes severe skin burns and eye damage
- H412 Harmful to aquatic life with long lasting effects
- H332 Harmful if inhaled
- H290 May be corrosive to metals
- P273 Avoid release to the environment
- P280 Wear protective gloves/protective clothing/eye protection/face protection
- P301+P330+P331 IF SWALLOWED: Rinse mouth. Do NOT induce vomiting
- P304+P340 IF INHALED: Remove person to fresh air and keep comfortable for breathing
- P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do - continue rinsing
- P310 Immediately call a POISON CENTER or doctor/physician
Formic acid:
- H331 Toxic if inhaled
- H319 Causes serious eye irritation
- H318 Causes serious eye damage
- H315 Causes skin irritation
- H314 Causes severe skin burns and eye damage
- H302 Harmful if swallowed
- H226 Flammable liquid and vapour
- P405 Store locked up.
- P403+P233 Store in a well-ventilated place. Keep container tightly closed.
- P370+P378 In case of fire: Use … for extinction.
- P337+P313 IF eye irritation persists: Get medical advice/attention.
- P305+P351+P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continuerinsing.
- P303+P361+P353 IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower.
- P280 Wear protective gloves/protective clothing/eye protection/face protection.
- P260 Do not breathe dust/fume/gas/mist/vapours/spray.
- P210 Keep away from heat/sparks/open flames/hot surfaces. — No smoking.
Trypsin
- H315 Causes skin irritation
- H319 Causes serious eye irritation
- H334 May cause allergy or asthma symptoms or breathing difficulties if inhaled
- H335 May cause respiratory irritation
- P261 Avoid breathing dust/fume/gas/mist/vapors/spray
- P264 Wash thoroughly after handling
- P271 Use only outdoors or in a well-ventilated area
- P272 Contaminated work clothing should not be allowed out of the workplace
- P280 Wear protective gloves/protective clothing/eye protection/face protection
- P302 + P352 IF ON SKIN: wash with plenty of water
- P304 + P340 IF INHALED: Remove person to fresh air and keep comfortable for breathing
- P305+ P351+ P338 IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses if present and easy to do - continue rinsing
- P332+P313 IF SKIN irritation occurs: Get medical advice/attention
- P310 Immediately call a POISON CENTER or doctor/physician
- P403+P233 Store in a well-ventilated place. Keep container tightly closed
- P501 Dispose of contents/container to an authorised landfill
Before start
1. Reagents should be prepared and stored in glass labware only. We recommend using the same glass labware for every analysis to avoid external contamination by detergents or other chemicals
2. We recommend preparing reagents just before starting the protocol.
3. Handle reagents under a fume hood only
4. Use ultrapure water for the preparation of reagents
5. Wipe down all working benches with70% isopropyl alcoholContributed by users or 70% Ethanol Contributed by users prior to begin working
6. All steps should be performed in a dedicated wet chemistry laboratory. If the presence of human remains is suspected, we recommend sampling in a DNA-free/P3 laboratory
Initial sampling
Initial sampling
15m
15m
Sample extraction
Use a pair of plyers to break apart a bone chip from bone sample or select a small bone fragment
Note
It is recommended to avoid drilling the specimens to obtain bone powder as the high speed of the drill head produces heat that can damage already degraded proteins
Note
To avoid unnecessary damage of archaeological specimens with no preserved proteins, a screening step using ATR-FTIR has been developed and will be published separately
Sample cleaning
Wipe the external surface of the bone with bleach10% BleachContributed by users
Remove the outer layer of the bone sample, either by polishing or manual removal
Sample homogenising
Grind manually with mortar and pestle until sample is homogenised into a fine powder
Sample weighing
Weigh out 20 mg - 30 mg bone powder for each technical/biological duplicate
Add bone powder to 1.5 mL LoBind eppendorf tube
Note
It is recommended to process technical duplicates (ideally triplicates) for each of the extracted samples
Decontamination
Place open tubes under UV light for at least 00:15:00
15m
Acid demineralisation
Acid demineralisation
4d 0h 15m
4d 0h 15m
Add 500 µL of freshly made 0.6 Molarity (M) Hydrochloric acidMerck MilliporeSigma (Sigma-Aldrich) to each sample
4d
Store samples at 4 °C until demineralisation is completed
Note
Demineralisation can take several days. When demineralised, bone becomes spongy and translucent
Monitor the samples to avoid protein loss
Note
If demineralisation stops (no bubbling is visible) but the samples are not fully demineralised, exchange supernatant after centrifugation at 10000 x g, Room temperature, 00:10:00
Note
Prepare laboratory blanks at this step. Blanks should follow the same steps as the archaeological samples
When samples are demineralised, centrifuge samples at 10000 x g, Room temperature, 00:10:00 to precipitate the extracted proteins to the base of the tube
Extract supernatant and discard. If the acid soluble fraction wants to be analysed, store supernatant in a new tube at -20 °C
Note
Do not disturb bone pellet during pipetting
10m
Acid washing
Wash bone pellet with 500 µL of 8 50 millimolar (mM) Ammonium bicarbonate (NH4HCO3)Merck MilliporeSigma (Sigma-Aldrich)Catalog #AC393212500 Centrifuge at 10000 x g, Room temperature, 00:05:00 Discard buffer
Repeat the washing step until samples reach neutral pH
Equipment
Centrifuge
NAME
Benchtop Centrifuge
TYPE
Eppendorf
BRAND
5405000441
SKU
LINK
Any benchtop centrifuge will suffice
SPECIFICATIONS
5m
Protein denaturation
Protein denaturation
1h 10m
1h 10m
Buffer exchange
Add 100 µL of 50 millimolar (mM) AmBic to bone pellet
Gelatinization
Incubate samples for 01:00:00 at 65 °C
Note
Samples will be hot! Take care when handling the samples
Equipment
ThermoMixer
NAME
Benchtop Incubator
TYPE
Eppendorf
BRAND
5382000023
SKU
LINK
Any heat block will suffice
SPECIFICATIONS
1h
Centrifuge samples at 10000 x g, Room temperature, 00:10:00
10m
Prepare two 1.5 mL LoBind Eppendorf tubes for each sample
Note
Label tubes with sample name/number followed by 'A' and 'B'
Transfer 50 µL of supernatant to each of the labelled 1.5 mL LoBind Eppendorf tubes
Note
If required, store samples that will not be use at -20 °C
Protein content quantification
Protein content quantification
To calculate the amount of protease required to successfully digest your sample into peptides, use the protein assay method more suited for your samples and your plate reader
Note
Commercial protein assay kits provide user guides which describe the working procedures specific for each kit. In general, and based in our experience, BCA kits perform well for archaeological samples with low collagen/protein concentrations
Protein digestion (trypsin)
Protein digestion (trypsin)
1h
1h
Note
There are several available Mass Spectrometry Grade Proteases, each following specific digestion procedures. Here we describe the common protocol used forPierce Trypsin ProteaseThermo Fisher ScientificCatalog #90057
Reconstitute lyophilised trypsin with 50 millimolar (mM) Glacial acetic acidFisher ScientificCatalog #A38-500 to a concentration of 1 mg/mL
Add protease to a ratio of 1:20-1:100
Incubate samples Overnight at 400 rpm, 37°C
1h
Stop digestion by freezing at -20 °C
Concentrate samples to dryness
Note
Samples can be concentrated in a Speedy Vacuum
Concentration can take around 06:00:00 , depending on equipment, temperature, volume and other factors. Monitor samples carefully
Peptide purification (C18 tips, Agilent OMIX)
Peptide purification (C18 tips, Agilent OMIX)
10m
10m
Preparation
Prepare conditioning, washing and elution solution (see Materials for details on reagents)
Label three 0.5 mL tubes per sample. Each tube will contain conditioning solution (40 µL ), washing solution (70 µL ) and elution solution (30 µL ). Add an additional tube per sample for solvent waste
Pretreat sample
Resuspend samples in 20 µL 0.1 % (v/v) Trifluoroacetic acid (TFA)Bio Basic Inc.Catalog #TC8960.SIZE.100mL .
Vortex thoroughly and sonicate for 00:10:00 to ensure resuspension
10m
Condition tip
Set pipettor at 10 µL and securely attach the tip
Aspirate 10 µL of conditioning solution and discard solvent. Repeat. Keep pipette plunger depressed.
Equilibrate tip
Aspirate 10 µL washing solution and discard. Repeat. Keep pipette plunger depressed.
Bind sample
Aspirate up to 10 µL of pre-treated sample into tip. Dispense and aspirate for up to 10 cycles to improve binding. Dispense sample into a waste tube. Keep pipette plunger depressed
Purify/desalt
Aspirate 10 µL of washing solution and discard solvent. Repeat 3 times. Keep pipette plunger depressed.
Elute
Elute with10 µL 0.1 % (v/v) Formic acid, LC-MS gradeThermo Fisher ScientificCatalog #28905 in75 % (v/v) Acetonitrile (ACN)Fischer ScientificCatalog #10660131 . Dispense in glass vial LC/MS-grade
Concentrate samples in Speedy Vacuum until dry
Peptide injection into LC MS/MS
Peptide injection into LC MS/MS
10m
10m
Resuspend samples in 30 µL of 5 % (v/v) ACN in 0.1 % (v/v) Formic Acid
Sonicate for 00:10:00 and mix thoroughly making sure there are no bubbles on the bottom of the vial insert
Note
Samples are now ready to be injected into the LC MS/MS instrument. If not analysing immediately, store samples in 4 °C (1-2 days) or -20 °C (long storage)
10m