Assessment of a Protease-Free Method for Body Fluid Identification in Sexual Assault Evidence by Targeted...

Area of Science:

• Forensic Science
• Analytical Chemistry
• Biochemistry

Background:

• Traditional serological screening for body fluid identification in sexual assault cases lacks sensitivity and specificity compared to DNA profiling.
• Current molecular approaches like mass spectrometry (MS)-based proteomics are hindered by complex workflows and specialized equipment.

Purpose of the Study:

• To develop a streamlined, protease-free assay for rapid identification of saliva and seminal fluid in sexual assault evidence.
• To overcome limitations of existing methods by utilizing targeted DDA on a Q Exactive MS with a simplified workflow.

Main Methods:

• A single-step extraction followed by targeted data-dependent acquisition (DDA) on a Q Exactive MS with a 25-min separation.
• Analysis using Byos software in a 96-well plate format for potential automation.
• Discovery analysis of 50 saliva and 60 semen samples, followed by synthesis of peptide standards for targeted assay development.

Main Results:

• Identification of 7 saliva biomarkers (PRB1, PRB2, PRB4, PRH1, STATH, HTN1, SMR3B) and 5 seminal fluid biomarkers (SEMG1, SEMG2, PSA, PAP, PIP).
• Successful validation of the targeted assay using 168 forensic casework-type samples.
• Demonstrated robustness with diluted, laundered, and environmentally challenged samples on various substrates.

Conclusions:

• The developed protease-free assay provides a sensitive and specific method for identifying saliva and seminal fluid in forensic samples.
• This streamlined approach enhances throughput and is suitable for automation, addressing a critical need in sexual assault investigations.
• The method offers a viable alternative to traditional assays, improving the analysis of challenging forensic evidence.