Direct seminal fluid identification by protease‐free high‐resolution mass spectrometry

Serological screening of sexual assault evidence has traditionally focused on enzyme activity and immunochromatographic assays that provide only a presumptive indication of seminal fluid and have limited sensitivity relative to DNA testing. Seminal fluid detection based on protein mass spectrometry represents a “Next Gen” serological technology that overcomes the specificity and sensitivity limitations of traditional serological screening but requires time‐consuming sample preparation protocols. This paper describes a novel “peptidomics” approach to seminal fluid detection that eliminates the need for lengthy trypsin digestion. This streamlines sample preparation to a one‐step process followed by high‐resolution mass spectrometry to identify naturally occurring seminal fluid peptides and low‐molecular weight proteins. Multiple protein biomarkers of seminal fluid were consistently and confidently identified based on the multiplexed detection of numerous endogenous peptides. These included Semenogelin I and II (90% and 86% sequence coverage, respectively); Prostate Specific Antigen/p30 (29% sequence coverage); and Prostatic Acid Phosphatase (24% sequence coverage). The performance of this streamlined peptidomics approach to seminal fluid identification in a forensic context was also assessed using simulated casework samples of the type typically collected as part of a sexual assault examination (e.g., oral and vaginal swabs stained with semen). The resulting data demonstrate that sub‐microliter quantities of seminal fluid on cotton swabs can be recovered and reliably detected. This supports the forensic applicability of a peptidomic assay for seminal fluid identification with same‐day sample preparation and analysis. Future development and streamlined multiplex peptidomic assays for additional biological stains can easily be envisaged.