Mineral-bound ostrich eggshell peptides survive for up to 3.8 Ma in equatorial Africa
Claims for exceptional preservation of biomolecules in the fossil record are contested. Here we demonstrate the role of surface stabilisation in significantly prolonging protein sequence survival to ~3.8 million years. The intracrystalline environment of calcite ostrich (Struthionidae) eggshell encapsulates uterine proteins and molecular dynamics simulations of struthiocalcin-1 & -2, the dominant proteins within the eggshell, reveal that they bind to the mineral surface in distinct domains. By ~3.8 million years the struthiocalcin-1 domain with the lowest calculated binding energy is selectively preserved in eggshell samples from equatorial Africa. Sequence survival is explained by entropy loss of the peptide and water, lowering the effective temperature of the local environment at the peptide mineral interface.
Sample Processing Protocol
Ostrich eggshell samples were ground and bleached for 72 hours (NaOCl 12% wt/vol) and rinsed thoroughly before demineralization. Amino acid and mass spectrometry analysis of ancient proteins was conducted using published techniques (Crisp et al, 2013; Buckley et al, 2009). Modifications include the use of trypsin and elastase as digestion enzymes for separate preparations (Welker et al, 2015). Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses were performed on Thermo Scientific Orbitrap platforms in two laboratories: 1) Advanced Proteomics Facility, Target Discovery Institute, Nuffield Department of Medicine, University of Oxford; 2) Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen.
Data Processing Protocol
Resulting spectra were searched against the Struthioniformes genomes using PEAKS (version 7.5 (30)). For PEAKS, FDR rate was set at 0.5%, with proteins accepted with -10lgp scores ≥ 40 and ALC (%) ≥ 80. All modifications were searched using the SPIDER function of PEAKS.