Forensic analysis of human tissues and body fluids has historically relied on serological tests and microscopy for body fluid identification, and short tandem repeat (STR) analysis for human identification. These approaches remain central to forensic biology, but they can be limited when evidence is environmentally degraded, naturally keratinized, or when the question requires source attribution rather than only donor attribution. Proteins provide a complementary molecular target because peptide bonds are chemically robust, and some structural proteins can persist under conditions that damage other biomolecules such as DNA. In addition, nonsynonymous single nucleotide polymorphisms can be expressed as genetically variant peptides (GVPs), allowing protein sequence information to contribute to human identification, while body-fluid-specific peptides (BFPs) can support body fluid identification. Forensic proteomics therefore addresses questions adjacent to conventional DNA typing such as what tissue or body fluid is present, can a DNA-poor sample still yield genetically informative evidence, and how robust are these signals after environmental insult?
At the National Institute of Standards and Technology (NIST), current work is evaluating the practical recovery and robustness of proteomic information from human hair and semen. For hair, recent method development has focused on trace keratinized material: direct extraction from a single 5 cm shaft coupled with optimized single-pot, solid-phase-enhanced sample preparation (SP3) increased protein and GVP detection, improved keratin and keratin-associated protein sequence coverage, and was approximately three times faster than the previous in-gel workflow. Related studies are evaluating digestion strategies, including non-specific enzymes, and the effect of common cosmetic treatments on hair chemistry and GVP detection. For semen, NIST is comparing STR, GVP, and BFP persistence after environmental challenges. Semen proteomes were analyzed using a dialysis-based workflow, while paired DNA analysis used the EZ1 DNA Investigator Kit, Quantifiler Trio, and PowerPlex Fusion 6C. STR profiles showed no apparent degradation after seven days at 70 °C or 24 hours of ultraviolet irradiation. Proteomics identified 34 robust GVPs and 31 robust BFPs after heat exposure, 49 GVPs and 40 BFPs after ultraviolet irradiation, and 5 GVPs and 10 BFPs after three months of uncontrolled outdoor exposure. More persistent proteins included SEMG1 and SEMG2, whereas more degradation-prone proteins included MUC5B and PIGR. These results support the utility of proteomics as a robust alternative for human identification and body fluid identification.
The New York City Office of Chief Medical Examiner (NYC OCME) provides a casework-focused example of forensic proteomics moving from discovery research into operational implementation. The program progressed from broad proteomic discovery and multiplex body-fluid identification toward a targeted liquid chromatography-tandem mass spectrometry molecular serology assay for confirmatory identification of blood, saliva, and semen. The operational assay uses redundant peptide-marker panels, transition-based detection, retention-time concordance, fragment-ion ratio scoring, peak-area criteria, formal quality-control metrics, Skyline review, and analyst-facing interpretation rules. This design is important for forensic use because the assay is intended to classify whether a target body fluid is present, not to estimate the quantity of fluid deposited. Validation work included neat and mock samples, mixtures, non-target human fluids, non-human materials, aged samples, and donor-to-donor biological variation. Reported performance was strongest when sufficient protein was present and more limited at very low protein loads, an important boundary condition for casework interpretation. The assay was accredited by ANAB, approved by the New York State Commission on Forensic Science for casework use, and introduced into NYC OCME casework in 2023, with public validation and procedural materials subsequently released. The broader NYC OCME research program also includes menstrual blood and vaginal-fluid marker discovery, species attribution from proteomic sequence information, informatics approaches for mixtures and difficult samples, and probabilistic interpretation of sparse collagen datasets, showing how the same analytical framework can extend beyond conventional serology targets.
Together, these studies show that forensic proteomics is progressing from discovery toward practical forensic interpretation. Its near-term value is not as a replacement for STR typing, but as a complementary framework for body-fluid identification and human identification when DNA is not detected, in low abundance, or degraded. This transition is now being formalized through the recently formed Organization of Scientific Area Committees for Forensic Science (OSAC) Forensic Proteomics Task Group, which is developing standards and guidance for quality, training, analytical methods, interpretation, reporting, and testimony across mass spectrometry-based body-fluid, taxonomic, and human-identification applications.
Forensic analysis of human tissues and body fluids has historically relied on serological tests and microscopy for body fluid identification, and short tandem repeat (STR) analysis for human identification. These approaches remain central to forensic biology, but they can be limited when evidence is environmentally degraded, naturally keratinized, or when the question requires source attribution rather than only donor attribution. Proteins provide a complementary molecular target because peptide bonds are chemically robust, and some structural proteins can persist under conditions that damage other biomolecules such as DNA. In addition, nonsynonymous single nucleotide polymorphisms can be expressed as genetically variant peptides (GVPs), allowing protein sequence information to contribute to human identification, while body-fluid-specific peptides (BFPs) can support body fluid identification. Forensic proteomics therefore addresses questions adjacent to conventional DNA typing such as what tissue or body fluid is present, can a DNA-poor sample still yield genetically informative evidence, and how robust are these signals after environmental insult?
At the National Institute of Standards and Technology (NIST), current work is evaluating the practical recovery and robustness of proteomic information from human hair and semen. For hair, recent method development has focused on trace keratinized material: direct extraction from a single 5 cm shaft coupled with optimized single-pot, solid-phase-enhanced sample preparation (SP3) increased protein and GVP detection, improved keratin and keratin-associated protein sequence coverage, and was approximately three times faster than the previous in-gel workflow. Related studies are evaluating digestion strategies, including non-specific enzymes, and the effect of common cosmetic treatments on hair chemistry and GVP detection. For semen, NIST is comparing STR, GVP, and BFP persistence after environmental challenges. Semen proteomes were analyzed using a dialysis-based workflow, while paired DNA analysis used the EZ1 DNA Investigator Kit, Quantifiler Trio, and PowerPlex Fusion 6C. STR profiles showed no apparent degradation after seven days at 70 °C or 24 hours of ultraviolet irradiation. Proteomics identified 34 robust GVPs and 31 robust BFPs after heat exposure, 49 GVPs and 40 BFPs after ultraviolet irradiation, and 5 GVPs and 10 BFPs after three months of uncontrolled outdoor exposure. More persistent proteins included SEMG1 and SEMG2, whereas more degradation-prone proteins included MUC5B and PIGR. These results support the utility of proteomics as a robust alternative for human identification and body fluid identification.
The New York City Office of Chief Medical Examiner (NYC OCME) provides a casework-focused example of forensic proteomics moving from discovery research into operational implementation. The program progressed from broad proteomic discovery and multiplex body-fluid identification toward a targeted liquid chromatography-tandem mass spectrometry molecular serology assay for confirmatory identification of blood, saliva, and semen. The operational assay uses redundant peptide-marker panels, transition-based detection, retention-time concordance, fragment-ion ratio scoring, peak-area criteria, formal quality-control metrics, Skyline review, and analyst-facing interpretation rules. This design is important for forensic use because the assay is intended to classify whether a target body fluid is present, not to estimate the quantity of fluid deposited. Validation work included neat and mock samples, mixtures, non-target human fluids, non-human materials, aged samples, and donor-to-donor biological variation. Reported performance was strongest when sufficient protein was present and more limited at very low protein loads, an important boundary condition for casework interpretation. The assay was accredited by ANAB, approved by the New York State Commission on Forensic Science for casework use, and introduced into NYC OCME casework in 2023, with public validation and procedural materials subsequently released. The broader NYC OCME research program also includes menstrual blood and vaginal-fluid marker discovery, species attribution from proteomic sequence information, informatics approaches for mixtures and difficult samples, and probabilistic interpretation of sparse collagen datasets, showing how the same analytical framework can extend beyond conventional serology targets.
Together, these studies show that forensic proteomics is progressing from discovery toward practical forensic interpretation. Its near-term value is not as a replacement for STR typing, but as a complementary framework for body-fluid identification and human identification when DNA is not detected, in low abundance, or degraded. This transition is now being formalized through the recently formed Organization of Scientific Area Committees for Forensic Science (OSAC) Forensic Proteomics Task Group, which is developing standards and guidance for quality, training, analytical methods, interpretation, reporting, and testimony across mass spectrometry-based body-fluid, taxonomic, and human-identification applications.
Workshop currently at capacity. A waitlist is available to join on our registration page.
Health Scientist in the Mass Spectrometry Data Center Group, National Institute of Standards and Technology
Zachary Goecker is a Health Scientist in the Mass Spectrometry Data Center Group at the National Institute of Standards and Technology. His research applies proteomic mass spectrometry to forensic biology, with an emphasis on genetically variant peptides for human identification and body-fluid-specific peptides for body fluid identification.
Principal Scientist, Department of Forensic Biology, New York City Office of Chief Medical Examiner
Donald Siegel is Principal Scientist in the Department of Forensic Biology at the New York City Office of Chief Medical Examiner, where he leads the Emerging Technologies Group. His work focuses on evaluating and implementing emerging forensic technologies, including proteomic mass spectrometry and massively parallel sequencing, for forensic casework applications.
