Bone Proteomics In Forensic Investigations: Advances, Challenges, And Methodological Innovations

Similarly to their forensic application, for which bone remains are an invaluable source of biological information, traditional analytical techniques, mainly DNA analysis, frequently lacks the sensitivity to overstep the challenges with aged or environmentally challenged material. Bone proteomics – the mass analysis of proteins preserved in skeletal tissue – has become a powerful complementary tool, taking advantage of the longer-term durability of proteins in comparison with nucleic acids. Here, we have consolidated the state of the art of forensic bone proteomics concentrating on important developments, persisting challenges and major methodological refinements. Bone proteome analyses for PMI, age-and sex determination and species determination have shown a rapid progress focusing on the analysis for certain post-translational modifications of proteins (e.g. Biglycan deamidation) or protein markers (e.g. Fetuin-A, Amelogenin, COL1A2). Nevertheless, some challenges exist for the application of the field. These challenges include the intricate interplay of diagenetic factors affecting bone composition, and of diverse environmental conditions (temperature, pH, humidity, burial context) and recycling of skeletal proteins on survival, exogenous proteome mixtures, and endogenous proteome signals, biological inherent variability among individual organisms, and robust bioinformatic approaches for analysis of often low-abundant or modified peptides. Moreover, inter-comparison and implementation of these methods are difficult due to the absence of standardized, validated protocols among different laboratories. Recent methodological developments, like improved protein extraction protocols (S-Trap workflow for instance), novel MS acquisition strategies (as data independent acquisition approach DIA) and complex bioinformatics pipelines, are attempting to overcome these issues. Future work, including biomarker validation, pathway elucidation, contamination control, workflow optimization and forensic protein databases work are required to translate the potential of bone proteomics to practical and robust tools for forensic investigations.