Paleoproteomics on Eggshells: A Comparative Study of Extraction Methods and Techniques

Palaeoproteomics is a promising technique for studying ancient archaeological and paleontological materials, offering valuable taxonomic, evolutionary, and ecological insights. Among biological substrates, eggshells serve as an excellent source of peptides due to their mineralized structure, which facilitates long-term protein preservation. However, the efficiency of protein extraction depends on the protocols and techniques employed, making it essential to optimize methodologies to maximize peptide recovery while minimizing contamination. This thesis compares different protein extraction protocols for eggshell, evaluating their efficiency in both qualitative and quantitative terms. Various approaches were tested, including demineralization with hydrochloric acid (HCl) and salts of ethylenediaminetetraacetic acid (EDTA), filtration using magnetic microspheres (SeraMag SpeedBead Hydrophilic and Hydrophobic) and ultrafilters (0.4 mL capacity, 3 kDa molecular weight cut-off, MWCO), and non-filtration methods. Additionally, purification was performed with either “stage tips” produced in our laboratory under non-sterile conditions using 200 μl pipette tips and a C-18 disk or pre-packaged C-18 tips (Zip Tips). Blank experiments were also conducted to identify primary sources of contamination, such as plastic residues and keratin. Data on peptide recovery from each experimental set was obtained through mass spectrometry analysis (matrix assisted laser desorption ionization-time of flight, MALDI-TOF). We selected a list of eggshell-specific peptide markers, which are visualized as “peaks” on MALDI-TOF spectra, and compared our results by quantifying three parameters: manually identified peaks, software-detected peaks, and ubiquity of each peak across the aliquots. This revealed differences between the protocols: demineralization with HCl resulted in slightly higher peptide recovery compared to EDTA, while EDTA demonstrated better recovery for lower-mass peptides. Among the filtration methods, ultrafiltration resulted in higher peptide detection across the spots (ubiquity). In the purification step, Stage Tips yielded better protein recovery when paired with HCl, whereas ZipTips performed more effectively in EDTA-treated samples that were not filtered. Finally, blank experiments confirmed the presence of modern contaminants, likely originating from laboratory reagents and consumables, emphasizing the need for contamination-reduction strategies. The results of this study provide key insights into optimized protein extraction protocols for eggshell, enhancing their application in paleontological and archaeological research.