Saliva identification in forensic samples by automated microextraction and intact mass analysis of statherin.

The enzyme α-amylase has long been a commonly targeted protein in serological tests for saliva. While being especially abundant in saliva, α-amylase is detectable in vaginal secretions, sweat, fecal matter, breast milk and other matrices. As a result, assays for α-amylase only provide a presumptive indication of saliva. The availability of mass spectrometry-based tools for the detection of less abundant, but more specific, protein targets (e.g., human statherin) has enabled the development of high confidence assays for human saliva. Sample throughput, however, has traditionally been low due to multi-step workflows for protein extraction, quantitation, enzymatic digestion, solid phase cleanup, and nano-/capillary-based chromatography. Here, we present two novel "direct" single-stage extraction strategies for sample preparation. These feature immunoaffinity purification and reversed-phase solid-phase microextraction in conjunction with intact mass analysis of human statherin for saliva identification. Mass analysis was performed on the Thermo Scientific Q-Exactive™ Orbitrap mass spectrometer with a 10-min analytical run time. Data analysis was performed using Byos® from Protein Metrics. Two sample sets were analyzed with a population of 20 individuals to evaluate detection reliability. A series of casework-type samples were then assayed to evaluate performance in an authentic forensic context. Statherin was confidently identified in 92% and 71% of samples extracted using the immunoaffinity purification and solid phase microextraction approaches, respectively. Overall, immunoaffinity purification outperformed the solid phase microextraction, especially with complex mixtures. In toto, robotic extraction and intact mass spectrometry enable the reliable identification of trace human saliva in a variety of sample types.

High-throughput seminal fluid identification by automated immunoaffinity mass spectrometry.

The identification of semen during a criminal investigation may be a critical component in the prosecution of a sexual assault. Commonly employed enzymatic and affinity-based methods for detection lack specificity, are time-consuming, and only provide a presumptive indication that semen is present where microscopic visualization is unable to meet the throughput demands. Contrary to traditional approaches, protein mass spectrometry provides true confirmatory results, but multiday sample preparation and nanoflow sample separation requirements have limited the practical applicability of these approaches. Aiming at streamlining sexual assault screening by mass spectrometry, the work here coupled a 60-minute rapid tryptic digestion, semenogelin-II peptide affinity purification on an Agilent AssayMap Bravo automation platform, and a 3-minute targeted LC-MS/MS method on an Agilent 6495 triple quadrupole mass spectrometer operating in multiple reaction monitoring mode for detecting semenogelin-II peptides in sexual assault samples. The developed assay was assessed using casework-type samples and was successful in detecting trace levels (0.0001 µl) of semen recovered from both cotton and vaginal swabs, as well as semen recovered from vaginal swabs during menses or adulterated with personal lubricants. This work represents a promising technique for high-throughput seminal fluid identification in sexual assault-type samples by mass spectrometry.

Developmental validation of a multiplex proteomic assay for the identification of forensically relevant biological fluids.

The aim of this study was to validate a multiplex proteomic assay for the identification of high-specificity protein biomarkers by multiple reaction monitoring mass spectrometry on a triple quadrupole mass spectrometer for the accurate, reliable, and confirmatory identification of bodily fluids commonly encountered in a forensic context. This includes the identification of peripheral blood, semen, saliva, urine, and vaginal/menstrual fluid. The assay is able to efficiently identify pure or mixed stains through the identification of target peptide fragments originating from tissue-specific proteins including: uromodulin from urine; prostatic acid phosphatase, prostate specific antigen and semenogelin-II for semen; statherin, submaxillary gland androgen-regulated protein 3B and amylase for saliva; cornulin, martrigel-induced gene C4 protein, suprabasin and neutrophil gelatinase-associated lipocalin for vaginal/menstrual fluid; and alpha-1 antitrypsin, hemopexin, and hemoglobin subunit beta for peripheral blood. Based on the results of the developmental validation studies which included an assessment of reproducibility and repeatability, sensitivity, species specificity, carryover, mixtures, as well as a series of casework type samples. Only a small selection of case samples was unable to unambiguously identify the target fluid including urine recovered from substrates as well as semen when mixed with personal lubricants. Overall, the mass spectrometry-based workflow offers significant advantages compared to existing serological methods.

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.

Effects of organic acids and common household products on the occurrence of false positive test results using immunochromatographic assays.

Forensic serological analyses often rely on lateral flow immunochromatographic assays to detect proteins that are characteristic of forensically relevant body fluids. In this study, we demonstrate that a positive result, however, is not limited to target protein binding. Citric and lactic acids at various pH levels were tested using 9 different commercial immunochromatographic assays. Varying rates of false positive results were observed with commercial serological assays irrespective of brand or target biological fluid over a wide pH range. The use of kit specific buffers were only partially effective in mitigating the occurrence of organic acid-associated false positive results. Common household products containing organic acids were also tested and found to produce non-specific binding events. This is not to suggest that immunochromatographic assays are not useful as presumptive indicators of bodily fluids. Rather, this study provides a cautionary demonstration of the ease with which organic acids in common household products can generate false positives results. This finding underscores the presumptive nature of these antibody-based lateral flow assay systems.

A survey of senior practitioners regarding most desirable qualifications for hiring and advancement within forensic science.

In today's environment in the field of forensic science where continual advancements in technology and analytical approaches are the norm, the need for forensic practitioners with more specialized and subject-specific knowledge is critical. An updated survey targeting the preferred educational requirements by senior practitioners, crime laboratory directors and managers for entry level applicants was conducted. Results underscored a preference for specialized coursework within specific disciplines in preparing the next generation of forensic scientists while maintaining a strong foundation in the natural sciences at the undergraduate level. Practitioners, regardless of discipline, are seeking applicants with exposure to advanced curriculum content in addition to refined professional skills and critical thinking capabilities. The results of this survey reflect a transition in the needs of crime laboratory employers from a general, broad based criminalistics curriculum as described under current accreditation guidelines, to a focused subject matter rich curriculum with additional management and professional content.