Morphology of the dog neonate and estimation of its age at death

As the deciduous first molar does not erupt while the deciduous canine and deciduous second molar are erupting in the maxilla, the age at death of 2017HA1016 was estimated at 2 weeks after birth, based on the Mori’s reference chart26 (Fig. 1). Most elements of the skeleton were preserved, and the recovered bones of the skeleton are described in Supplementary Table 1.

Figure 1 Photo of the main bones of neonatal dog skeleton (2017HA1016): (1) maxilla and frontal; (2) parietal; (3) scapula R; (4) humerus L; (5) radius R; (6) tibia R; (7) femur R. Full size image

Detected proteins

Only collagen α-1(I) fragments (COL1A1) were detected in the fish bone by searching against the dog proteome, and only actin cytoplasmic 1 (ACTB) and COL1A1 were detected with ≥2 non-overlapping peptides in the soil sample. The following results and discussion are therefore limited to the data obtained from the analysis of the bone fragments of the 2017HA1016 dog. Protein groups identified in blanks but not in the dog bones with ≥2 peptides were not considered.

A total of 200 protein groups were detected across the EDTA and pellet fractions of the rib and vertebra bones (Supplementary Table 2). A total of 143 protein groups (71.5%) were detected in both the EDTA and the pellet fractions, and 154 protein groups (77.0%) were retrieved from both rib and vertebra. Recovered proteomes can differ in multiple experimental fractions extracted from a single sample32 and from different bone elements from a single individual33. PANTHER analysis indicated that 51.6% of protein groups are classified as an extracellular component (Supplementary Table 3) and 41.3% are related with binding function (Supplementary Table 4). Mean deamidation rates of samples were 36.3 ± 4.8% for asparagine (N) and 11.0 ± 2.1% for glutamine (Q) (n = 8; Supplementary Table 5). Deamidation of glutamine and asparagine residues is a non-enzymatic modification that occurs over time, resulting in a + 0.98402 Da mass shift caused by the direct or indirect hydrolysis of the N and Q side-chain amide group34.

Biological marker proteins

Protein groups characteristic of fetuses or newborns were detected from bones of 2017HA1016 (Table 2). Alpha-fetoprotein (AFP), a major plasma protein expressed in yolk sac and fetal liver and only detected in fetuses or young neonates35, was found in all of the four bones. AFP may play a role comparable to that of serum albumin in the adult, and its concentration is at its highest just after delivery (14080 ± 5944 μg/mL) and rapidly decreases during the first 2 weeks after birth (70.21 ± 52.92 μg/mL) in dogs35.

Table 2 Characteristic protein groups for fetus or growing infant and milk that were detected in 2017HA1016. Full size table

Several protein groups that are related with endochondral bone formation were detected (Table 2). Most bones, including ribs and vertebrae, develop via endochondral bone formation; endochondral cartilage templates are then replaced by calcified bone matrix36. Collagen type X, alpha-1 (COL10A1) has functions in bone formation, and is only expressed in endochondral cartilage that will be replaced by mature bone tissue in humans37. Epiphycan (EPYC) is a small leucine-rich proteoglycan that is mostly found in fetal and neonatal epiphyseal cartilage in mice, bovines, and chickens38. EPYC has important roles in cartilage development and its maintenance39. Detection of these proteins (Table 2) is consistent with the age at death of the 2017HA1016 individual and it confirms that palaeoproteomics can retrieve growth- or developmental stage-specific proteins from archaeological bone remain28,40.

In addition, two milk proteins were detected from the EDTA fractions of two distinct subsamples, i.e. the distal and proximal ends of a single rib. Two peptides of beta-lactoglobulin-1 (LGB1) were detected from the EDTA fraction of the distal part of 2017HA1016’s rib (Fig. 2, Table 3). LGB1 is a major whey protein, absent in some mammalian species, including humans41. Although its exact physiological function is not determined yet, LGB1 binds several hydrophobic ligands and thus may act as specific transporters41.

Figure 2 MS2 spectra of peptides assigned to LGB1. Full size image

Table 3 Matched peptides for milk proteins. Full size table

Protein BLAST searches indicated that the two detected peptides of LGB1 sequences were identical to those of dogs with the highest score. One of the detected peptide sequences (TLEVDNEVMEK) also matches the sequence of glycodelin of Vulpes vulpes (red fox). The identification of the other sequence (TMEDLDLQK) is supported by a spectrum including the complete y-ion series (Fig. 2), however, it’s quality is lower, most probably due to random co-fragmentation of the precursor with another peptide. This spectrum could also be assigned equally confidently to the deamidated (N → D) peptide sequence of LGB from Callorhinus ursinus (northern fur seal), Odobenus rosmarus divergens (Pacific walrus), and Leptonychotes weddellii (Weddell seal). However, its assignment to dog LGB1 represents the most parsimonious interpretation.

In addition, two peptides of whey acidic protein (WAP) were detected from EDTA fraction of the proximal part of 2017HA1016’s rib (Fig. 3, Table 3). WAP is a major whey protein present in dog milk, which seems to play important roles in regulating the proliferation of mammary epithelial cells42. The detected peptides of WAP perfectly match with the dog WAP sequence reported by Seki and colleagues42. Protein BLAST searches indicated that the sequences recovered uniquely match dog WAP. Unassigned higher peaks in MS2 spectrum of CCLSVCAMR (Fig. 3) were mostly originated from neutral losses (Supplementary Fig. 2).

Figure 3 MS2 spectra of peptides assigned to WAP. Full size image

Since the neonatal dog is too young to secrete breast milk, the detected peptides of LGB1 and WAP would most probably originate from its mother. The alternative origin of LGB1 and WAP from the above-mentioned non-dog species is archaeologically and ecologically highly unlikely. The possibility of contamination is excluded because no LGB1 and WAP peptides were detected in the negative controls. Finally, the only species that matches all observed peptides is Canis lupus familiaris.

We also detected protein groups that are expressed in milk, but also in other tissues. Milk fat globule-EGF factor 8 (MFGE8) facilitates scavenging of the dying cells from the tissue and is an essential factor for controling the progression of various inflammatory diseases43. MFGE8 is a component of milk fat globule membrane protein, but it is also expressed ubiquitously in other cells and tissues43. Fourteen peptide-spectrum matches of MFGE8 were obtained from the dog bone samples.