Tiny Hopes─Assessing Protein Preservation in Collagen-Depleted Bones

Understanding human evolution relies on biomolecular data from ancient skeletal tissues, yet warm climates often cause complete collagen loss, excluding many regions from the study. This research investigates the survival of non-collagenous proteins (NCPs) and low-molecular-weight proteins in archaeological bones deemed collagen-free by traditional metrics. Using a multi-method approach, we employed sandwich enzyme-linked immunosorbent assays for osteocalcin quantification, Qubit fluorometry for total protein, and liquid chromatography tandem mass spectrometry (LC-MS/MS) for characterization, complemented by Fourier transform infrared spectroscopy to assess the diagenetic state. Samples included collagen-depleted bones from Neolithic Lebanon and Palaeolithic France, with well-preserved controls from Neolithic Serbia and Paleolithic Russia. Results indicate that bone-associated NCPs, including osteocalcin, survive only if the insoluble collagen is preserved. Methodologically, tangential flow filtration outperformed centrifugal devices for protein recovery. EDTA demineralization with FASP was most effective for maximizing collagen recovery, while HCl demineralized protein precipitation best detected unique NCPs. Collagen was identified in the soluble supernatants of most collagen-depleted bones. Abundant collagen peptides were identified in a sample with a 0% collagen yield and a very low amide-to-phosphate ratio. These findings demonstrate that bones unsuitable for traditional dating can still retain measurable collagen, broadening the range of biomolecular analyses possible in warm and humid climates.