Insights into the Differential Preservation of Bone Proteomes in Inhumed and Entombed Cadavers from Italian Forensic Caseworks

Abstract

Bone is a hard biological tissue and a precious reservoir of information in forensic investigations as it retains key biomolecules commonly used for identification purposes. Bone proteins have recently attracted significant interest for their potential in estimating post-mortem interval (PMI) and age at death (AAD). However, the preservation of such proteins is highly dependent on intrinsic and extrinsic factors that can hinder the potential application of molecular techniques to forensic sciences. The present study aims at investigating the effects that two commonly used types of burial practices (entombment and inhumation) have on bone protein survival. The sample consists of 14 exhumed individuals from cemeteries in Southern Italy with different AADs (29-85 years) and PMIs (1-37 years). LC-MS/MS analyses show that 16 proteins are better preserved under the entombed conditions and 4 proteins are better preserved under the inhumed conditions, whereas no clear differences are detected for post-translational protein modifications. Furthermore, several potential "stable" protein markers (i.e., proteins not affected by the burial environment) are identified for PMI and AAD estimation. Overall, these results show that the two burial environments play a role in the differential preservation of noncollagenous proteins, confirming the potential of LC-MS/MS-based proteomics in forensic sciences.

Keywords: age-at-death; bone; burial environment; forensics; post-mortem interval; post-translational protein modifications.

Figure 1

Boxplot showing the four parameters evaluated with FTIR. Higher values are observed for the wooden coffin group (in blue) compared with the zinc-lined coffin group (in magenta) (reported, respectively, as “wood coffin” and “zinc coffin” in the figure).

Figure 2

PCA plot showing (A) the individual clustering according to the two burial conditions (blue dots for wooden coffins and magenta triangles for zinc-lined coffins, reported, respectively, as “wood coffin” and “zinc coffin” in the figure) and (B) the relative abundance of the 10 proteins that provided the highest contribution in the clustering.

Figure 3

Box plots showing the relative abundances of the 20 proteins that differed significantly (p < 0.05) between burial conditions. In blue (first boxes) are samples in the wooden coffin, and in magenta (second boxes) are samples in the zinc-lined coffin (reported as “zinc coffin” in the figure). Supplementary Table S1 provides the entire list of significant proteins according to the t-test.

Figure 4

STRING diagram of the proteins that shows the significant difference between the two burial conditions. The clusters show the presence of bone proteins (blue), collagen and collagen-binding proteins (red), also involved in bone metabolism, and plasma and blood proteins (green). NUCB2 is the only protein dissociated from the group. The line thickness indicates the strength of data support (edge confidence). K means clustering was used to visualize clusters (k = 4).

Figure 5

(A) PCA individuals plot showing the clustering according to the two burial conditions, wooden (in blue) and zinc-lined (in magenta) coffins (reported, respectively, as “wood coffin” and “zinc coffin” in the figure), and (B) the contribution of the six deamidated peptides to the PCA.

Figure 6

Boxplots showing the deamidation rates for specific peptides and associated proteins (in the title of each boxplot) in wooden (in blue) and zinc-lined (in magenta) coffins (reported, respectively, as “wood coffin” and “zinc coffin” in the figure). No significant differences were found between any of the unmodified and modified peptides.

Figure 7

Error plots of the protein abundances showing a significant negative correlation with both age and PMI when n = 28 samples are included in the calculation. The first seven plots show the relationship with age, whereas the second seven plots show the correlation of the same protein relative abundances with PMI. Correlation coefficients and significance can be found in Supplementary Table S2.

Figure 8

Scatterplot showing the relationship between the relative abundance of COCA1 and the chronological age of the individuals. PMIs are reported using the color code shown in the legend (blue for shorter PMIs, magenta for longer PMIs) and burial conditions (wooden and zinc-lined coffins, reported, respectively, as “wood coffin” and “zinc coffin” in the figure) with different shapes.