Mass spectrometry-based proteomic strategy for ecchymotic skin examination in forensic pathology

Abstract

Mass spectrometry (MS)-based proteomics has recently attracted the attention from forensic pathologists. This work is the first report of the development of a shotgun bottom-up proteomic approach based on rapid protein extraction and nano-liquid chromatography/high-resolution mass spectrometry applied to full-thickness human skin for the differential analysis of normal and ecchymotic tissues to identify new biomarkers for bruise characterization and dating. We identified around 2000 proteins from each pooled extract. The method showed excellent precision on independent replicates, with Pearson correlation coefficients always higher than 95%. Glycophorin A, a known biomarker of vital wounds from immunochemical studies, was identified only in ecchymotic tissues, as confirmed by Western blotting analysis. This finding suggests that this protein can be used as a MS-detectable biomarker of wound vitality. By focusing on skin samples from individuals with known wound dating, besides Glycophorin A, other proteins differentially expressed in ecchymotic samples and dependant on wound age were identified, although further analysis on larger datasets are needed to validate these findings. This study paves the way for an in-depth investigation of the potential of MS-based techniques for wound examination in forensic pathology, overcoming the limitations of immunochemical assays.

Figure 1

(a) Home-made closed stainless-steel mortar; (b) Two-way ANOVA interaction plot for homogenization step. Concentration values expressed as µg protein/mg tissue.

Figure 2

Venn diagrams of proteins identified in ecchymotic (E) and normal (N) skin in the comparisons: (a) 1E versus 1N and 4E versus 4N replicates; (b) 2E versus 2N and 5E versus 5N replicates; (c) 3E versus 3N; (d) G0E versus G0N; (e) G1E versus G1N; (f) G0E versus G1E. A statistical evaluation was applied for each comparison, where applicable. Proteins were considered to be differentially expressed if they were present only in one condition or showed significant t-test difference (Student’s t-test FDR ≤ 0.05).

Figure 3

Venn diagram of the proteins identified by MS/MS exclusively in the ecchymotic skin in the comparison 1E versus 2E versus 3E versus 4E versus 5E versus G0E versus G1E.

Figure 4

(a) Cropped bands of WB probed with anti-Glycophorin A antibody (upper panel) of the extracts from ecchymotic (E) and normal (N) tissue pools from individuals 13–18. GAPDH was used as a loading control (lower panel) on the same samples. Original full-length blot images and both replicates used for the analysis are provided in Supplementary Fig. S3. (b) Densitometric analysis of the WB in the extracts from ecchymotic (grey) and normal (black) tissue pools. Data are mean ± standard deviation from two different gels. ***p < 0.001 ecchymotic versus normal skin.