1. Biochemistry and Chemical Biology
  2. Evolutionary Biology
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Protein sequences bound to mineral surfaces persist into deep time

  1. Beatrice Demarchi  Is a corresponding author
  2. Shaun Hall
  3. Teresa Roncal-Herrero
  4. Colin L Freeman  Is a corresponding author
  5. Jos Woolley
  6. Molly K Crisp
  7. Julie Wilson
  8. Anna Fotakis
  9. Roman Fischer
  10. Benedikt M Kessler
  11. Rosa Rakownikow Jersie-Christensen
  12. Jesper V Olsen
  13. James Haile
  14. Jessica Thomas
  15. Curtis W Marean
  16. John Parkington
  17. Samantha Presslee
  18. Julia Lee-Thorp
  19. Peter Ditchfield
  20. Jacqueline F Hamilton
  21. Martyn W Ward
  22. Chunting Michelle Wang
  23. Marvin D Shaw
  24. Terry Harrison
  25. Manuel Domínguez-Rodrigo
  26. Ross DE MacPhee
  27. Amandus Kwekason
  28. Michaela Ecker
  29. Liora Kolska Horwitz
  30. Michael Chazan
  31. Roland Kröger
  32. Jane Thomas-Oates
  33. John H Harding  Is a corresponding author
  34. Enrico Cappellini
  35. Kirsty Penkman
  36. Matthew J Collins  Is a corresponding author
  1. University of York, United Kingdom
  2. University of Sheffield, United Kingdom
  3. University of Copenhagen, Denmark
  4. University of Oxford, United Kingdom
  5. Bangor University, United Kingdom
  6. Arizona State University, United States
  7. Nelson Mandela Metropolitan University, South Africa
  8. University of Cape Town, South Africa
  9. New York University, United States
  10. Complutense University of Madrid, Spain
  11. American Museum of Natural History, United States
  12. National Museum of Tanzania, Tanzania
  13. The Hebrew University, Israel
  14. University of Toronto, Canada
  15. University of the Witwatersrand, South Africa
  16. Centre of Excellence in Mass Spectrometry, University of York, United States
Research Article
Cite this article as: eLife 2016;5:e17092 doi: 10.7554/eLife.17092
5 figures, 19 tables, 3 data sets and 4 additional files

Figures

Eggshell peptide sequences from Africa have thermal ages two orders of magnitude older than those reported for DNA or bone collagen.

(A) Sites reporting the oldest DNA and collagen sequences are from high latitude sites compared to ostrich eggshell samples from sites in Africa illustrated in (B) for which the current mean annual air temperatures are much higher. (C) Kinetic estimates of rates of decay for DNA (Lindahl and Nyberg, 1972), collagen (Buckley and Collins, 2011) and ostrich eggshell proteins (Crisp et al., 2013) were used to estimate thermal age assuming a constant 10°C (Figure 1—source data 1; Appendix 1. For Elands Bay Cave and Pinnacle Point the oldest samples are shown). Note log scale on the z-axis: struthiocalcin-1 peptide survival is two orders of magnitude greater than any previously reported sequence, offering scope for the survival of peptide sequences into deep time.

https://doi.org/10.7554/eLife.17092.003
Figure 1—source data 1

Data and calculations for thermal ages reported in Figure 1 and in Supplementary file 1.

https://doi.org/10.7554/eLife.17092.004
Figure 2 with 2 supplements
Proteome persistence and patterns of degradation.

(A) Amino acid racemization in ostrich eggshell up to 3.8 Ma old from sites in South Africa and Tanzania. (B) Linear increase of THAA Val D/L values with the log of thermal age. (C) Exponential decrease of the number of identified MS/MS spectra with age (THAA Val D/L). (D) The average hydropathicity of the peptides identified remains stable up to Val D/Ls ~1. Note that Val D/Ls > 1.0 are unexpected and may be due to decomposition processes occurring in the closed system. The intracrystalline proteome composition in modern eggshell does not vary across microstructural layers (Figure 2—figure supplement 1), but patterns of degradation are different between fossil samples and purified proteins degraded at high temperature in the absence of the mineral (Figure 2—figure supplement 2).

https://doi.org/10.7554/eLife.17092.005
Figure 2—figure supplement 1
Structure and composition of OES.

(A) modern (left) and fossil (LOT 13898; right) OES: crystalline (1), prismatic (2), cone (3) and organic (4) layers. (B) comparison of total THAA yields in each layer before and after bleaching. (C) comparison between the composition of bleached eggshell powder from the cone, palisade and crystalline layers.

https://doi.org/10.7554/eLife.17092.006
Figure 2—figure supplement 2
Proteome degradation.

(AB) fossil OES: (A) number of unique proteins; (B) mean peptide length (excluding contaminants). (CE) degradation of purified proteins in water: (C) number of unique proteins identified; (D) number of identified product ion spectra; (E) mean peptide length; (F) average hydropathicity. No peptides were detected in the 120 hr heated sample.

https://doi.org/10.7554/eLife.17092.007
Figure 3 with 2 supplements
Survival of struthiocalcin 1 and struthiocalcin 2 peptides.

Over time (and increasing THAA Val D/L values) the spectral count decreases as degradation progresses. Blue bars highlight the peptides investigated computationally (represented by the filled atoms in the models). Highly degraded samples (Val D/L ~0.8–1.1) preserve the DDDD-containing peptide. The full time series is shown for SCA-1 in Figure 3—figure supplement 1 and for SCA-2 in Figure 3—figure supplement 2.

https://doi.org/10.7554/eLife.17092.015
Figure 3—figure supplement 1
Frequency of identified spectra of SCA-1 in bleached OES (fossils) and purified proteins (kinetics).

Spectral count scale: 0–400 for fossil OES; 0–200 for kinetics. Sample 4605 has been recognized as burnt (Crisp, 2013) but excellent sequence preservation is observed. Low spectral counts for sample 4613 are likely due to sample preparation, as AAR did not identify this sample as problematic. Coloured bars show protein structure.

https://doi.org/10.7554/eLife.17092.016
Figure 3—figure supplement 2
Frequency of identified spectra of SCA-2 in bleached OES (fossils) and purified proteins (kinetics).

Spectral count scale: 0–400 for fossil OES; 0–200 for kinetics. Sample 4605 has been recognized as burnt (Crisp 2013) but excellent sequence preservation is observed. No SCA-2 peptides were found for sample 4613; this is likely due to sample preparation. Wonderwerk and Laetoli samples yielded some peptide sequence, but not consistently.

https://doi.org/10.7554/eLife.17092.017
Authenticity of the ancient sequences.

Amino acid analyses (A): Total concentrations in all eggshell samples (sum of Asx, Glx, Gly, Ala, Val and Ile). Carry-over: (B) Total ion chromatogram for one eggshell sample (EBC_1823) and the blank analysed immediately after (blank_post_EBC1823). (C) Spectral abundance of SCA-1 and SCA-2 in LC-MS/MS blanks. (D) SCA-1 coverage in the blank analysed after a Pinnacle Point eggshell sample PP_4652. Note 'DDDD-' and 'EEEED-' peptides and Asn deamidation. (E) Extracted ion chromatogram for LDDDDYPK in EBC_1823, blank_post_EBC1823 and EBC_1819. (F) Absolute and relative total abundance of 'DDDD' peptides in Laetoli samples/blanks. Signal reduction is at least 100-fold (more often 1000- or 10,000-fold). Independent replication and manual de novo sequencing of the peptides from Laetoli (Appendix 5, section A; Supplementary file 2), consistency of diagenesis-induced modifications (Appendix 5, section D; Supplementary file 3) and volatile organic compound analyses (Appendix 5, section E) were also used to validate the results obtained.

https://doi.org/10.7554/eLife.17092.018
Schematic diagram of energy barriers for peptide hydrolysis.

A pictorial representation of the energy barriers associated with the lysis of the peptide. The process in bulk water is depicted in red and the process at the surface is depicted in blue. The surface process shows a larger barrier due to the stabilization of the reactants at the surface.

https://doi.org/10.7554/eLife.17092.019

Tables

Table 1

Summary of archaeological and paleontological eggshell samples analysed in this study. See also Tables 37.

https://doi.org/10.7554/eLife.17092.008
SiteApproximate age range (ka)Approximate thermal age range (ka@10°C)Number of specimens
Elands Bay Cave0.3–160.5–458
Pinnacle Point 5/650–150120–4708
Wonderwerk~1000~36001
Olduvai~1340~163004
Laetoli2600–43008900–204003
Table 2

Binding of proteins to the (10.4) calcite surface. The binding energies calculated as (a) mean for the full protein (by minimization, see Appendix 3); (b) for four individual domains within the proteins (by molecular dynamics [ovocleidin]; by minimization [struthiocalcin]); (c) for the four domains treated as peptides (by molecular dynamics, see Appendix 3).

https://doi.org/10.7554/eLife.17092.009
OC-17SCA-1SCA-2
Charge on the protein+7 (balanced
by Cl-)
−11 (balanced by Na+, Ca2+)−10 (balanced byCa2+)
Binding energy (mean): kJ mol−1−197 ± 22−142 ± 33
Binding energy (domains): kJ mol−1−422 ± 43−423 ± 42 (YHHGEEEEDVWI)
−611 ± 44 (YSALDDDDYPKG)
−255 ± 72 (SDSEEEAGEEVW)
−231 ± 68 (ASIHSEEEHQAIV)
Binding energy (peptides only) kJ mol−1−142 ± 19 (YHHGEEEEDVWI)
−219 ± 24 (YSALDDDDYPKG)
−131 ± 32 (SDSEEEAGEEVW)
−122 ± 41 (ASIHSEEEHQAIV)
Water molecules displaced21.320.223.1
Estimated binding free energy: kJ mol−1−188 ± 37−159 ± 24−99 ± 39
Residence times
for water (ps)
[average surface bound water molecule = 120 ps]
130 ± 3 (YHHGEEEEDVWI)
135 ± 3 (YSALDDDDYPKG)
124 ± 4 (SDSEEEAGEEVW)
123 ± 5 (ASIHSEEEHQAIV)
Table 3

Summary of samples from Elands Bay Cave, South Africa. The stratigraphic layers have been independently dated by radiocarbon. Unpublished uncalibrated dates provided by J. Parkington. Date calibration was performed with OxCal v.4.2 (Ramsey, 2009. Calibration curves: IntCal13 for dates obtained on charcoal; Marine13 for dates obtained on shells/crayfish, DeltaR = 93 ± 28 [Dewar et al., 2012]). Age estimates for undated layers based on estimating the median (mid-point) of two dates obtained on layers bracketing the layer with OES samples.

https://doi.org/10.7554/eLife.17092.010
LOTNEaarLayerAge (cal BP) 95.4%Material used for 14C dating/notes
18686887Kaunda<323 (estimate)Layer above dates on layer NKOM
18726888George Best322–1008Layer between dates on layers NKOM and EDDI
18666889D. Lamour906–2282Layer between dates on layers EDDI and LARM
18496891Maroon Robson8773 ± 125Charcoal
18506893Nero8748–10096Layer between dates on layers Maroon Robson / Burnt Robeson
18236896Crayfish11545 ± 441Crayfish
18196899Smoke12589 ± 104Charcoal
18406907OBS 115208–15940Layer between dates on layers Smoke and SOSE
Table 4

Sample details for sub-fossil OES analysed for LC-MS/MS; from Pinnacle Point, South Africa. Stratigraphic information and weighted mean OSL age estimates (ka) for PP 5–6 (Karkanas et al., 2015) and PP 30 (Rector and Reed, 2010).

https://doi.org/10.7554/eLife.17092.011
SiteLOTNEaarArchaeological sample informationStratigraphic aggregateAge (ka)
PP5-646137676Plotted Find 102627, Lot 3151RBSR51 ± 2
PP5-646497283Plotted Find 165702, Lot 8038SGS64 ± 3
PP5-646717316Specimen 273467, Lot 3255SADBS71 ± 3
PP5-646057198Specimen 273489, Lot 3277SADBS71 ± 3
PP5-646527286Plotted Find 178331, Lot 8172ALBS72 ± 3
PP5-646757320Specimen 273514, Lot 7980LBSR81 ± 4
PP 3046837328Specimen 66008, Lot 1795Single horizon~151
PP 3046977342Specimen 65168, Lot 1750Single horizon~151
Table 5

Sample details for sub-fossil OES samples from Wonderwerk Cave, South Africa. Ages based on cosmogenic isotope burial dating and magnetostratigraphy, from Matmon et al. (2012).

https://doi.org/10.7554/eLife.17092.012
LOTNEaarStratumIndependent age (Ma)
1442610581ME46, SPF#4390, Exc. 1, stratum 10, square Q33, depth 15–20 cm1.07–0.99
Table 6

Sample details for fossil OES samples from Olduvai, Tanzania.

https://doi.org/10.7554/eLife.17092.013
LOTNEaarLocality/StratumIndependent age (Ma)
1557510955Sample BK09-31501.338 ± 0.024
1557810958Sample BK10-53091.338 ± 0.024
1557910959Sample BK09-26271.338 ± 0.024
1558210962Sample BK09-27061.338 ± 0.024
Table 7

Sample details for fossil OES samples from Laetoli, Tanzania. Ages of the strata and localities (40Ar/39Ar) from Deino (2011). LOT 13901 is attributed to Struthio camelus. LOTs 13902 and 13898 are attributed to Struthio kakesiensis (Harrison and Msuya, 2005).

https://doi.org/10.7554/eLife.17092.014
LOTNEaarLocality/StratumIndependent age (Ma)
1390110574Loc 15, Upper Ndolanya Beds~2.66
1390210573Loc 10 West, Upper Laetolil Beds~3.8–3.85
1389810575Kakesio 1−6, Lower Laetolil Beds~3.85 -> 4.3
Appendix 1—table 1

Historical temperature data. Weather station temperature data derived from the NOAA Baseline Climatological Dataset - Eischeid et al. (1995): The quality control of long-term climatological data using objective data analysis. Preprints of AMS Ninth Conference on Applied Climatology, Dallas, TX., January 15–20, 1995.

https://doi.org/10.7554/eLife.17092.024
WMO IDWmo station namelatitudelongitudeAlt. (m)MAT °C
7191700Eureka,N.W.T.79.98−85.9310−19.6
100800Svalbard Lufthavn78.2515.4727−6.3
420201Dundas Radio Greenland76.6−68.820−10.5
420200Thule A.B.76.52−68.577−12.1
7195702Fort Mcpherson67.4−134.930−9.3
7196501Fort Selkirk62.8−137.4454−3.9
358600Honington52.330.77549.6
3605801Ust-Kan50.9284.7510370.6
807500Burgos/Villafria42.373.6389410.2
6832800Tsabong−26.0522.4596020.14
6871200Cape Columbine−32.8317.856015.6
892800Mossel Bay (Cape St.)−34.1822.155917.7
Borehole data: data from NOAA Paleoclimatology Borehole Datasets http://www.ncdc.noaa.gov/paleo/borehole
CA-289-260.99−13415240
UK-STOWLANGTOFT52.280.85479.1
ES-ROMANERA37.69−7.3316620.2
ES-AC-1BILLITON37.6−6.8311018.7
ES-PB1ADAROVALVERDE37.56−6.7823718.6
TZ-LONGIDO−2.6136.47131624.3
TZ-BASOTU−4.3835.17173623.9
TZ-KIZAGA−4.4234.37147223.2
TZ-SIUYU−4.934.88167823.1
ZA-SB1−27.2825.5135719.2
ZA-AP11−28.321.0592823.7
ZA-PC227−29.3321.78105221.5
Appendix 1—table 2

Long term climate records: cores used in this study to assess the extent of depression at LGM (used to scale).

https://doi.org/10.7554/eLife.17092.025
RecordlatitudelongitudeMethodSST (°C) during LGMRef
ODP98258−16UK3713(Lawrence et al., 2009)
ODP88250.35−167.58UK379.1(Martínez-Garcia et al., 2010)
MD02-2594−33.3017.30Mg/Ca~16doi.pangaea.de/10.1594/PANGAEA.810663
MD962077−33.1731.25Mg/Ca~16doi.pangaea.de/10.1594/PANGAEA.810716
ODP722A16.6259.80UK3720.2(Herbert et al., 2010)
ODP662−1.39−11.74UK3718.7(Herbert et al., 2010)
ODP846−3.10−90.82Mg/Ca18.6(Herbert et al., 2010)
IODP114619.46116.27UK3724.3(Herbert et al., 2010)
Appendix 1—table 3

Kinetic parameters used for estimating thermal age.

https://doi.org/10.7554/eLife.17092.026
TargetChemical reactionActivation energy (kJ mol−1)Reference
Eggshell proteinsValine racemization117(Crisp et al., 2013)
DNADepurination123(Lindahl and Nyberg, 1972)
CollagenGelatinization173(Holmes et al., 2005)
Appendix 1—table 4

Thermal age calculations.

https://doi.org/10.7554/eLife.17092.027
SiteLatLongAlti (m)MAT (°C)ScaleΔT LGM (°C)RefAge (ka)RateEa(kJ)ln AMean thermal age (Ma@10°C)Min thermal age (Ma@10°C)Max thermal age (Ma@10°C)
Ellesmere Island78.55−82.5292−19.75.0−21(Ballantyne et al., 2010)3600Collagen173610.0030.0010.007
Ellesmere Island78.55−82.5292−19.75.0−21(Ballantyne et al., 2010)3800Collagen173610.0040.0010.011
Poolepynten7911.434−6.34.0−16(Hubberten, 2004)130DNA12741.20.0020.0020.002
Thistle Creek63.11−139.54483−4.11.0−4(Elias, 2001)560DNA12741.20.020.020.02
Thistle Creek63.11−139.54483−4.11.0−4(Elias, 2001)700DNA12741.20.030.030.03
Happisburgh52.821.5329.12.0−9(Harrison et al., 2015)700Collagen173610.220.190.25
Sima de los Huesos42.35−3.5210049.51.0−4(Moreno et al., 2012)400DNA12741.20.250.220.28
Elands Bay Cave−32.3218.321015.71.0−4(Martínez-Méndez, 2010)16Valine118380.040.030.05
Pinnacle Point−34.222.0920181.0−4(Bar-Matthews et al., 2010)150Valine118380.420.380.47
Wonderwerk27.823.55164518.60.6−2.5(Ecker et al., 2015)1000Valine118383.63.14.1
Olduvai−2.9935.35576260.6−2.5(Tierney et al., 2010) (Berke et al., 2012)1338Valine1183816.314.418.5
Laetoli−3.2235.19170018.70.6−2.5(Tierney et al., 2010) (Berke et al., 2012)3800Valine1183816.013.618.7
Appendix 2—table 1

THAA concentrations (pmol/mg) detected in OES microstructural layers; bleached and unbleached powders were analysed. Analytical errors measured on replicate analyses are < 5% (Asx = 3.80%; Glx = 3.81%; Gly = 4.89%; Ala = 3.67%; Val = 3.98%; Ile = 3.63%).

https://doi.org/10.7554/eLife.17092.028
[Asx][Glx][Ser][L-Thr][L-His][Gly][L-Arg][Ala][Tyr][Val][Phe][Leu][Ile]
Cone-
bleached
4925540939922589152069232655431415792815198342642280
Cone-
unbleached
1445217835134559140489419136810810234434297344334105007676
Palisade-
bleached
5990643444792347207966413427518315252768265950323052
Palisade-
unbleached
1001811545796745073667126916247908530524843450584465102
Whole-
bleached
6323653346622445203269763322531412632943273252893177
Whole-
unbleached
124121377499275420421614951722210670436460035342101526342
Crystalline-
bleached
45184181308513011399409719813528491704183832992225
Crystalline-
unbleached
2442224212179908100674823365123712005564459912107551907912535
Appendix 2—table 2

30 major protein groups identified in bleached modern OES (>20 unique peptides only).

https://doi.org/10.7554/eLife.17092.029
Protein groupAccession-lgPCoverage (%)#UniqueDescription
1gi|46396750344100346RecName: Full = Struthiocalcin-1; Short = SCA-1
2gi|4639675129398222RecName: Full = Struthiocalcin-2; Short = SCA-2
7gi|697501075, gi|67821762628139208Aggrecan core protein [Struthio camelus australis]
8gi|69750892426888126Vitelline membrane outer layer protein 1-like [Struthio camelus australis]
10gi|69750902926880113Serum albumin-like [Struthio camelus australis]
13gi|6974557832464089Tenascin isoform X3 [Struthio camelus australis]
11gi|6974772022347584Carbonic anhydrase 4 [Struthio camelus australis]
14gi|6974818282371582Mucin-5AC [Struthio camelus australis]
9gi|697523391, gi|6782215882286977Apolipoprotein D [Struthio camelus australis]
12gi|3751626482317576Immunoglobulin A heavy chain constant region secretory form partial [Struthio camelus]
15gi|697470179, gi|6974701772135163Mesothelin isoform X1 [Struthio camelus australis]
22gi|697433909, gi|6782065871954045Golgi apparatus protein 1 [Struthio camelus australis]
17gi|3751626441926342immunoglobulin M heavy chain constant region secretory form partial [Struthio camelus]
19gi|678209093, gi|6974411801976641Stanniocalcin-1 partial [Struthio camelus australis]
18gi|697514088, gi|6782198031978639Ovomucoid [Struthio camelus australis]
20gi|6974886111918939Serotriflin-like [Struthio camelus australis]
21gi|6782147781924238Delta and Notch-like epidermal growth factor-related receptor partial [Struthio camelus australis]
23gi|697475278, gi|697475274, gi|6974752801924137Polymeric immunoglobulin receptor [Struthio camelus australis]
26gi|6974309751813333BPI fold-containing family B member 4-like [Struthio camelus australis]
25gi|6974309361614232Uncharacterized protein LOC104140623 [Struthio camelus australis]
31gi|697485873, gi|6782148461726828Pigment epithelium-derived factor [Struthio camelus australis]
28gi|697430934, gi|6782057481533426BPI fold-containing family B member 4-like [Struthio camelus australis]
29gi|6974329181724126Prosaposin [Struthio camelus australis]
16gi|3751626541789624immunoglobulin lambda constant region partial [Struthio camelus]
30gi|697522911, gi|697522913, gi|6975229161725022Pantetheinase-like isoform X1 [Struthio camelus australis]
24gi|6974464191657122Beta-2-microglobulin [Struthio camelus australis]
27gi|678210026,gi|6782100251546322Cygnin [Struthio camelus australis]
34gi|6782163651733121Signal peptide CUB and EGF-like domain-containing protein 1 partial [Struthio camelus australis]
32gi|6974920531434221Ovalbumin {ECO:0000303|PubMed:21058653} [Struthio camelus australis]
37gi|6975056891471320Ovostatin-like [Struthio camelus australis]
Appendix 2—table 3

List of contaminant proteins detected in the 66 analyses, with total number of spectra identified per protein.

https://doi.org/10.7554/eLife.17092.030
Row labelsCount of #Spec
sp|TRYP_PIG|771
sp|K2C1_HUMAN|493
Chymotrypsin-like elastase family member 1 OS = Sus scrofa GN = CELA1 PE = 1 SV = 1422
sp|K1C9_HUMAN|301
sp|K1C10_HUMAN|216
sp|K22E_HUMAN|213
sp|TRFE_HUMAN|98
sp|ALBU_HUMAN|94
PREDICTED: keratin type II cytoskeletal cochleal isoform X1 [Struthio camelus australis]80
PREDICTED: keratin type II cytoskeletal cochleal isoform X2 [Struthio camelus australis]32
PREDICTED: keratin type II cytoskeletal 5-like [Struthio camelus australis]25
sp|RS27A_HUMAN|23
Keratin type II cytoskeletal 75 partial [Struthio camelus australis]20
sp|GFP_AEQVI|19
sp|TRY1_BOVIN|19
Keratin type II cytoskeletal cochleal partial [Struthio camelus australis]16
PREDICTED: keratin type II cytoskeletal cochleal isoform X3 [Struthio camelus australis]16
sp|ANT3_HUMAN|8
sp|HBB_HUMAN|8
Keratin type II cytoskeletal 75 [Struthio camelus australis]5
sp|TRFL_HUMAN|5
Keratin type I cytoskeletal 14 partial [Struthio camelus australis]3
PREDICTED: keratin type II cytoskeletal 4-like [Struthio camelus australis]3
sp|HBA_HUMAN|3
sp|K1C15_SHEEP|2
sp|GSTP1_HUMAN|1
Appendix 4—table 1

D/L values of archaeological OES samples analysed in this study. b = bleached; H* = THAA obtained by 24-hr acid hydrolysis; F = FAA. Ile A/I values could not be calculated for Laetoli samples due to the presence of a compound co-eluting with D-alloisoleucine. Analytical errors measured on replicate analyses are <5% (D/Ls: Asx = 0.54%; Glx = 1.18%; Ala = 3.94%; Val = 2.09%; Ile = 3.77%. Concentrations: Asx = 3.80%; Glx = 3.81%; Gly = 4.89%; Ala = 3.67%; Val = 3.98%; Ile = 3.63%). (★) Sample 4605 yielded high D/L values because this sample had been exposed to high temperatures in the burial environment (burning [Crisp, 2013]). Note: thermal age calculations were performed on the basis of the Hansen model (Hansen et al., 2013); due to the absence of more continuous record for younger (last 2000 years) samples in the Hansen record, the Elands Bay Cave time points <1600 years refined using the SST record from core MD02-2594 - Dyez14 (Dyez et al. 2014).

https://doi.org/10.7554/eLife.17092.031
LOTNEaarAsx D/LGlx D/LAla D/LVal D/LIle A/IThermal age (years)
18686887bH*0.2910.0610.0660.0750.052401−564
18686887bF0.3460.0740.1190.0980.076
18726888bH*0.2680.0560.0610.0250.0321313–1932
18726888bF0.2120.0790.1000.0000.000
18666889bH*0.2780.0570.0660.0280.0523962−5704
18666889bF0.3310.0620.1160.0000.076
18496891bH*0.4140.0950.1420.0530.08119,759–27,951
18496891bF0.5550.1360.2260.1380.169
18506893bH*0.5240.1410.2150.0800.13319,759–27,951
18506893bF0.6980.1770.3680.1660.254
18236896bH*0.4620.1170.1850.0730.09125,657–36,249
18236896bF0.6210.1610.3010.1580.208
18196899bH*0.7140.2220.3170.1120.18426,878–37,877
18196899bF0.8060.2940.4420.2170.332
18406907bH*0.4690.1230.1870.0950.12232,379–44,863
18406907bF0.6650.1710.3270.1680.241
4605(★)7198bH*0.8660.7170.8400.5250.708166,094–198,795
4605(★)7198bF0.9310.7820.9310.6920.955
46137676bH*0.6610.2260.4110.2100.269122,139–148,223
46137676bF0.8000.3450.5700.3490.472
46497283bH*0.7120.3140.5000.2550.320151,169–181,807
46497283bF0.8460.3800.7290.4280.590
46527286bH*0.6780.2770.4630.2330.302168,899–202,088
46527286bF0.8220.3550.6980.3910.528
46717316bH*0.6810.2700.4680.2420.299166,094–198,795
46717316bF0.8630.3390.6870.3840.577
46757320bH*0.6870.2790.4940.2790.330192,147–230,607
46757320bF0.8320.3210.6880.4230.590
46837328bH*0.7640.3920.6760.3730.505378,398–467,602
46837328bF0.8790.4250.8210.5230.737
46977342bH*0.7520.3880.6530.3680.497378,398–467,602
46977342bF0.9040.6590.9200.7770.973
1442610581bH*0.7300.8700.8550.8550.9853,238,624–4,188,207
1442610581bF0.7900.9300.9601.0101.280
1557510955bH*0.8601.0350.9811.001>1.214,387,543–18,460,416
1557510955bF0.9241.0010.9611.005>1.2
1557810958bH*0.8911.0400.9841.007>1.214,387,543–18,460,416
1557810958bF0.9161.0090.9600.999>1.2
1557910959bH*0.8111.0210.9651.007>1.214,387,543–18,460,416
1557910959bF0.9261.0060.9691.005>1.2
1558210962bH*0.8811.0330.9761.012>1.214,387,543–18,460,416
1558210962bF0.9151.0020.9471.016>1.2
1390210573bH*0.9651.0500.9251.12>1.213,764,246–18,893,425
1390210573bF0.9351.0300.9251.065>1.2
1390110574bH*0.9201.0400.9451.16>1.28,943,148–11,841,107
1390110574bF0.9351.0150.9301.08>1.2
1389810575bH*0.9451.0500.9301.17>1.214,746,875–20,367,942
1389810575bF0.9351.0100.9301.095>1.2
Appendix 4—table 2

%FAA values (%FAA = [FAA]/[THAA] * 100). b = bleached. Total% FAA for Laetoli are calculated on the basis of Asx, Gly, Ala, Val only. * [Ala] and [Gly] > 100% are likely due to the effect of decomposition of other amino acids to FAA Gly and FAA Ala (e.g. Ser) (Walton, 1998). (★) Sample 4605 had been exposed to high temperatures in the burial environment (burning) (Crisp 2013).

https://doi.org/10.7554/eLife.17092.032
LOTNEaarAsxGlyAlaValIleAverage
186868877161819613
1872688841110326
18666889183539151324
18496891264141211830
18506893171457343030
18236896364147232134
18196899224556312836
18406907184051262332
4605(★)7198707483615669
46137676535766444152
46497283494963403948
46527286484761424048
46717316506172474557
46757320625974484758
46837328907191646676
46977342558396645871
144261058150122*138*908497
155751095553475546n/a50
155781095870627262n/a67
155791095929273126n/a28
155821096278627464n/a69
139021057374648265n/a71
139011057488789274n/a83
138981057587769373n/a82
Appendix 4—table 3

: Proteins identified in the purified extract from bleached OES, heated at 140°C in ultrapure water for 2, 8, 24 and 120 hr (identified by at least 2 unique peptide sequences). Values are number of peptide sequences identified.

https://doi.org/10.7554/eLife.17092.033
Protein description2 hr8 hr24 hr120 hrTotal
RecName: Full = Struthiocalcin-1; Short = SCA-129521316
RecName: Full = Struthiocalcin-2; Short = SCA-21248132
Vitelline membrane outer layer protein 1-like [Struthio camelus australis]22224
von Willebrand factor partial [Struthio camelus australis]2020
immunonoglobulin heavy chain variable region partial [Struthio camelus]1818
Apolipoprotein D [Struthio camelus australis]14216
Mitogen-activated protein kinase MLT [Struthio camelus australis]7916
Aggrecan core protein [Struthio camelus australis]1313
iron binding protein [Struthio camelus]99
Histone H4 [Struthio camelus australis]448
Cyclin-K [Struthio camelus australis]77
BPI fold-containing family B member 4 partial [Struthio camelus australis]426
Histone H2B 1/2/3/4/6 [Struthio camelus australis]336
Complement C3 [Struthio camelus australis]235
PREDICTED: histone H2B 1/2/3/4/6 [Struthio camelus australis]224
PREDICTED: polymeric immunoglobulin receptor [Struthio camelus australis]33
Carbonic anhydrase 4 partial [Struthio camelus australis]22
Appendix 5—table 1

Peptides identified in all Laetoli OES samples, prepared in York (analysed in Oxford) and Copenhagen. (*Gla) = this residue was found to be in the decarboxylated form, i.e. glutamate residues that have been post-translationally modified by vitamin K-dependent carboxylation to form gamma-carboxyglutamic acid (Gla), which binds calcium. We conducted manual de novo analyses of all product ion spectra and determined either complete or partial sequences for all of them, independently identifying sequences assigned (assisted de novo) by the software (PEAKS Studio).

https://doi.org/10.7554/eLife.17092.034
SequenceProteinLaboratoryScoreMS/MS count
AGAHLASIHTSEEHRSCA-1Copenhagen47.144
HYSALDDDDYPKGKSCA-1Copenhagen35.692
AGAHLASIHSCA-1Copenhagen31.453
ERNAFICKSCA-1Copenhagen28.121
GNCYGYFRSCA-1Copenhagen28.11
DVWIGLFRSCA-1Copenhagen26.385
ALDDDDYPKSCA-1York39.3928
ALDDDDYPKGSCA-1York41.3414
DDDDYPKGKSCA-1York40.793
DDDYPKGKSCA-1York32.891
HYSALDDDDYPKSCA-1York51.091
KHYSALDDDDYPKSCA-1York34.862
LDDDDYPKSCA-1York34.3512
LDDDDYPKGSCA-1York35.36
LDDDDYPKGKSCA-1York35.663
SALDDDDYPKSCA-1York41.0410
SALDDDDYPKGSCA-1York39.155
YSALDDDDYPKSCA-1York34.463
YSALDDDDYPKGSCA-1York31.93
RAEAWCRSCA-1York30.651
CYGFFPQELSWRSCA-2Copenhagen30.981
KPFICEYRTSCA-2Copenhagen25.031
GE(*Gla)EVWIGLHRPLGRSCA-2York37.332
LDYGSWYRSCA-2York35.11
AGE(*Gla)EVWIGLHRPLGRSCA-2York34.642
Appendix 5—table 2

Crushed eggshell sulfur-containing VOC emissions from 2.7 Ma OES (Laetoli LOT 13901). NA = no structural isomer can be determined.

https://doi.org/10.7554/eLife.17092.035
CompoundRetention time (min)Measured m/zChemical formulaNIST MS similarityReverse fit
Ethane thiol/dimethylsulfide3.4262.0196C2H6SNANA
Methylthioethane5.5976.0354C3H8S726821
Diethylsulfide8.2490.0511C4H10S725834
1-methylthiopropane8.6390.0509C4H10S646721
Unknown isomer8.86104.0663C5H12SNANANA
2-ethylthio-propane9.66104.0664C5H12S535723
2-methyl-1-(methylthio)-propane10.26104.0670C5H12S636798
ethylpropylsulfide10.85104.0671C5H12S628801
2-methyl-2-(methylthio)-butane11.89118.0823C6H14S567777
1-(ethylthio)-2-methyl-propane12.18118.0824C6H14S817856
2-methyl-3-(methylthio)-butane12.286118.0823C6H14S600820
Unknown isomer12.791118.0826C6H14SNANA
3-methyl-1-(methylthio)-butane12.821118.0827C6H14S779872
Tetrahydro-2,5-dimethylthiophene13.06116.0670C6H12S657741
Tetrahydro-2,5-dimethylthiophene13.14116.0670C6H12S622707

Data availability

The following data sets were generated
  1. 1
    Mineral-bound ostrich eggshell peptides survive for up to 3.8 Ma in equatorial Africa
    1. Demarchi B
    (2016)
    Publicly available at EBI PRIDE (accession no. PXD003786).
  2. 2
    BioProject ID PRJNA314978
    1. Haile J
    (2016)
    Publicly available at NCBI BioSample (accession no: SAMN04546447).
  3. 3
    Simulations on calcite -protein interactions
    1. Harding J
    2. Hall S
    3. Freeman C
    (2016)
    Publicly available at The University of Sheffield figshare (https://sheffield.figshare.com/).

Additional files

Supplementary file 1

Survival of ostrich eggshell proteins in time.

The proteins identified in each ostrich eggshell sample are reported, together with the number of identified peptides and the percentage coverage, Val D/L value and hydropathicity.

https://doi.org/10.7554/eLife.17092.020
Supplementary file 2

Product ion spectra.

Raw spectra (manually annotated on the basis of PEAKS assignments) of all the identified sequences identified in panel 1. 2–9: Copenhagen dataset; 10–26: York/Oxford dataset.

https://doi.org/10.7554/eLife.17092.021
Supplementary file 3

Diagenesis-induced modifications.

Modifications detected in SCA-1 and SCA-2 sequences in all OES samples analysed.

https://doi.org/10.7554/eLife.17092.022
Supplementary file 4

Full proteomics dataset.

This Excel file reports all the peptide and protein data for ostrich eggshell samples and the blanks.

https://doi.org/10.7554/eLife.17092.023

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