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

McKiernan, Heather E.; Danielson, Phillip B.; Brown, Catherine O.; Signaevsky, Masha; Westring, Christian G.; Legg, Kevin M.

doi:10.1016/j.forsciint.2021.110908

Cited by 8 publications

(11 citation statements)

References 33 publications

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“…The reported detection of statherin protein in a blood plasma protein reference dataset compiled nearly a decade and a half ago raises similar concerns [30]. However, it should be stated that statherin has not been detected in blood in any modern forensic discovery, verification, or developmental validation studies [17, 27, 31]. Moreover, no study has utilized certified reference material to confirm the presence of the statherin protein by empirically comparing the MS1 and MS2 spectra as well as retention times between the reference material and identification in blood.…”

Section: Resultsmentioning

confidence: 99%

“…Statherin has been identified in multiple discovery proteomic studies of human saliva, measured in specific applications to oral health, and has been routinely utilized for saliva identification as a highly specific mRNA marker and as marker for both targeted and untargeted mass spectrometry [24][25][26][27]. Therefore, statherin has been utilized as a reliable and specific biomarker for the identification of human saliva in applications with clear forensic utility.…”

Section: Statherin Characterization and Confirmationmentioning

confidence: 99%

“…The sensitivity with which such additional markers can be detected was illustrated in a previously published study [27] using a bottom-up protocol on a nanoflow triple quadrupole MS operating in MRM mode. In that study, it was possible to reliably detect submaxillary gland androgen-regulated protein 3B in 97 nL of saliva.…”

Section: Performance Assessmentmentioning

confidence: 99%

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

Brown,

Westring,

Danielson

et al. 2024

Journal of Forensic Sciences

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Statherin Characterization and Confirmationmentioning

confidence: 99%

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

Brown,

Westring,

Danielson

et al. 2024

Journal of Forensic Sciences

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“…In response to these limitations, several confirmatory technologies for semen identification have been developed using RNA-based methods [6][7][8], epigenetics [9,10], raman spectroscopy [11,12], and protein mass spectrometry [13][14][15][16][17][18]. Of note, protein identification by mass spectrometry (MS) has been shown to be a sensitive and specific method for the identification of several semen-specific protein biomarkers including semenogelin I/II, prostate-specific antigen, and prostatic acid phosphatase [14,17].…”

Section: Highlightsmentioning

confidence: 99%

High‐throughput seminal fluid identification by automated immunoaffinity mass spectrometry

Davidovics

Saw

Brown

et al. 2022

Journal of Forensic Sciences

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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.

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“…By detecting multiple peptides in a single assay from more than one protein per fluid, the assay can be considered not only confirmatory but also human-based and suitable for mixtures. Sensitive and specific assays have been developed including blood, semen, seminal fluid, saliva, vaginal/ menstrual fluid and urine [277][278][279][280] using methods such as matrix-assisted laser desorption/ionization (MALDI) MS [277], quadrupole time-of-flight MS [278,279] MALDI MS Profiling and Imaging [281] and MALDI time-of-flight MS [282] from samples as diverse as blood-stained finger marks [281] and blood and vaginal cellular material under fingernails [282], although difficulties in identifying urine recovered from substrates and semen samples that had been mixed with lubricants were found [280]. A challenge to be addressed is that the protein content of each fluid/ tissue varies considerably, and whilst it is possible to detect very small amounts of blood in saliva, the opposite was not true [277][278][279].…”

Section: Proteomementioning

confidence: 99%

On the Identification of Body Fluids and Tissues: A Crucial Link in the Investigation and Solution of Crime

Sijen

Harbison

2021

Genes

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Body fluid and body tissue identification are important in forensic science as they can provide key evidence in a criminal investigation and may assist the court in reaching conclusions. Establishing a link between identifying the fluid or tissue and the DNA profile adds further weight to this evidence. Many forensic laboratories retain techniques for the identification of biological fluids that have been widely used for some time. More recently, many different biomarkers and technologies have been proposed for identification of body fluids and tissues of forensic relevance some of which are now used in forensic casework. Here, we summarize the role of body fluid/ tissue identification in the evaluation of forensic evidence, describe how such evidence is detected at the crime scene and in the laboratory, elaborate different technologies available to do this, and reflect real life experiences. We explain how, by including this information, crucial links can be made to aid in the investigation and solution of crime.

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

Cited by 8 publications

References 33 publications

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

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

High‐throughput seminal fluid identification by automated immunoaffinity mass spectrometry

On the Identification of Body Fluids and Tissues: A Crucial Link in the Investigation and Solution of Crime

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