Bloodstain Proteomics as a Molecular Clock for Estimating Time Since Deposition, Biological Age, and Sex in Forensic Investigations

Abstract

Blood is one of the most informative biological traces recovered from crime scenes. Beyond conventional DNA profiling, its proteome may provide contextual information concerning when a stain was deposited and broad biological characteristics of the donor. This critical review examines three interconnected applications of forensic blood proteomics: time since deposition estimation, biological-age estimation and biological-sex estimation. We evaluate evidence from haemoglobin oxidation, plasma ageing signatures and sex-associated proteins; examine environmental, substrate and donor-related confounders; and compare the statistical and machine-learning approaches used for prediction. Six proteins, ITIHS, HSPA9, SNAP91, FIL, XPO4 and NCF2, currently provide the strongest replicated feature-selection evidence for age association in dried bloodstains. We further propose a nanoLC-timsTOF diaPASEF workflow for future validation, combining trapped-ion mobility, rigorous quality control, and multi-output models to simultaneously estimate several forensic attributes. Although a unified assay has not yet been validated in a single representative bloodstain cohort, the available evidence supports further development of proteomics as a complement to DNA profiling for crime-scene reconstruction.