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.

在性侵害案件调查中，包括免疫侧向流检测（immunological lateral flow assays）在内的血清学筛查仍是体液识别的常用手段，但其灵敏度与特异性均不及现代DNA分型技术。为填补这一技术空白，学界已探索包括基于质谱（MS）的蛋白质组学在内的替代分子检测方法，但此类方法因自下而上（bottom-up）流程冗长、依赖科研级仪器设备而难以推广应用。本研究报道了一种简便高效、无需蛋白酶处理的检测方法，用于性侵害物证中的唾液与精液识别。研究采用单步提取法处理案件类型体液样本，通过Q Exactive质谱（Q Exactive MS）上的靶向数据依赖性采集（targeted DDA）模式进行分析，色谱分离时长为25分钟，并借助Byos软件完成数据检索。所采用的96孔板体系适配高通量自动化操作。发现数据集共包含50份唾液样本与60份精液样本（其中5份来自输精管结扎男性的样本）。通过分析最终鉴定出7种唾液生物标志物（PRB1、PRB2、PRB4、PRH1、STATH、HTN1与SMR3B）以及5种精液生物标志物（SEMG1、SEMG2、PSA、PAP与PIP）。研究合成了肽段标准品以验证发现阶段的结果，并据此开发靶向检测方法。本研究使用168份法医案件类型样本对该方法进行了验证，涵盖不同载体上的稀释、洗涤及受环境胁迫的样本。