Human hair shafts are frequently encountered biological evidence at crime scenes. However, routine STR profiling is often unsuccessful because nuclear DNA in hair shafts is highly degraded. In contrast, hair proteins are chemically stable and retain genetic information in the form of single amino acid polymorphisms (SAPs), which can be detected as genetically variant peptides (GVPs). However, most existing studies remain at the stage of discovery proteomics, and there is still a lack of robust, high-sensitivity targeted detection strategies and multiplex marker systems suitable for practical forensic applications.

In this study, we developed a multiplex peptide panel for forensic individual identification based on SAPs detected in the human hair shaft proteome. Deep-coverage proteomic analysis was performed on hair shafts from 110 individuals. The number of identified proteins ranged from 628 to 2620, with a median of 1462.5, a mean of 1463, and a coefficient of variation (CV) of 0.28. The number of detected peptides ranged from 3936 to 13,647, with a median of 7616, a mean of 7626, and a CV of 0.21, demonstrating robust and deep proteome coverage across individuals.

A total of 2516 SAP loci were identified in the cohort using protein database containing East Asian population’s SAPs. By integrating SAP detection with exome genotype frequency data, the median cumulative random match probability (RMP) for hair shaft-based identification reached 3.92 × 10⁻¹¹, ranged from 9.02×10-7 to 1.90×10-2, indicating strong discrimination power. From these candidates, 46 GVPs corresponding to 23 informative SAP loci were selected to construct a targeted multiplex panel. The parallel reaction monitoring (PRM) targeted proteomic method was further developed for sensitive and reproducible detection of these markers. Compared with SAP genotyping derived from discovery proteomics, the PRM approach significantly improved the detection rate and accuracy of SAP typing. Notably, one GVP in the KRTAP10-5 protein contained two SAP loci that formed a haplotype defined by rs7509970 and rs464424. This haplotype exhibited good individual discriminative power and population variation.

Overall, the multiplex peptide panel and PRM workflow enhance the reliability of hair shaft proteomic genotyping and represent a critical step toward the practical forensic application of SAP-based individual identification from hair shaft.

Authors

• Lei Feng (Key Laboratory of Forensic Genetics of Ministry of Public Security, Institute of Forensic Science, Ministry of Public Security, China)
• Dongsheng Ding (Key Laboratory of Forensic Genetics of Ministry of Public Security, Institute of Forensic Science, Ministry of Public Security, China)
• Zhiyuan Yang (Key Laboratory of Forensic Genetics of Ministry of Public Security, Institute of Forensic Science, Ministry of Public Security, China)