Mass spectrometry-based proteomics in forensic investigations: a focused review of LC-MS applications

Abstract Background Proteomics, the large-scale study of proteins, has long been a vital tool in biological research. Its extension into forensic science, termed forensic proteomics, has opened new avenues for evidence analysis due to its high specificity and diverse applicability. Proteins, being stable and sample-rich, provide valuable insights in various forensic contexts, enhancing both identification and investigative accuracy. Main body Forensic proteomics has demonstrated promising applications in individual differentiation, ethnic and sex identification, biological age estimation, determination of the post-mortem interval (PMI), and biomarker-based identification of bodily fluids and tissues. It also plays a critical role in anti-doping efforts by detecting illicit peptides, proteins, and hormones. Commonly analyzed human samples include hair, bone, muscle, blood, and fingerprints. Among the many forensic challenges, PMI estimation remains one of the most debated and in need of methodological improvement. Despite its growing relevance, the field requires further validation and standardization to ensure reproducibility and legal acceptance of proteomic evidence. Conclusion This review offers a comprehensive overview of forensic proteomics, highlighting current methodologies, human sample-based applications, and future prospects. By addressing existing challenges and exploring emerging directions, it aims to support forensic investigators and researchers in adopting proteomics as a reliable tool for evidence analysis and interpretation. Key points: Forensic proteomics enables individual profiling by identifying ethnicity, sex, biological age, body fluids, tissues, and PMI using protein signatures from samples such as bone, hair, blood, and fingermarks. Advanced mass spectrometry approaches, including tandem MS, top-down, middle-down, and bottom-up proteomics, enhance the sensitivity and specificity of forensic investigations. Biomarker-driven analysis is emphasized, with key proteins such as feutin A (age estimation), biglycan (PMI), and keratin peptides (sex and ethnicity) offering valuable forensic insights. Challenges in forensic proteomics include sample degradation, lack of methodological standardization, and species differentiation, which limit reproducibility and legal admissibility. The review supports current findings about adopting forensic proteomics as a reliable and admissible tool in legal and investigative contexts.