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.