Assessment of a Protease-Free Method for Body Fluid Identification in Sexual Assault Evidence by Targeted High-Resolution Mass Spectrometry

Serological screening, including immunological lateral flow assays, remains common for body fluid identification in sexual assault investigations but lacks the sensitivity and specificity of modern DNA profiling. To address this gap, alternative molecular approaches, including MS-based proteomics, have been explored. However, adoption is hindered by lengthy bottom-up workflows and reliance on research-grade instrumentation. Here, a streamlined, protease-free assay for the identification of saliva and seminal fluid in sexual assault evidence is described. Casework-type body fluid samples were extracted in a single step and analyzed by targeted DDA on a Q Exactive MS with a 25-min separation and data search using Byos software. The 96-well plate format used is amenable to higher-throughput automation. Discovery data sets included 50 saliva and 60 semen samples (including samples from 5 vasectomized males). This resulted in the identification of 7 saliva biomarkers (PRB1, PRB2, PRB4, PRH1, STATH, HTN1, and SMR3B) and 5 seminal fluid biomarkers (SEMG1, SEMG2, PSA, PAP, and PIP). Peptide standards were synthesized to confirm the discovery results and to develop a targeted assay. The method was successfully validated using 168 forensic casework-type samples, including diluted, laundered, and environmentally challenged samples on a variety of substrates.