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

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

doi:10.1016/j.forsciint.2021.110908

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

Forensic Sci Int. 2021 Jul 17:326:110908. doi: 10.1016/j.forsciint.2021.110908. Online ahead of print.

Authors

Heather E McKiernan¹, Phillip B Danielson², Catherine O Brown², Masha Signaevsky¹, Christian G Westring³, Kevin M Legg⁴

Affiliations

¹ Department of Chemistry and Physics, Arcadia University, Glenside, PA 19038, USA.
² The Center for Forensic Science Research & Education, Willow Grove, PA 19090, USA; The University of Denver, Department of Biological Sciences, Denver, CO 80208, USA.
³ Purdue University Northwest, Center for Crime, Forensics, and Security Analysis, Hammond, IN 46323, USA.
⁴ The Center for Forensic Science Research & Education, Willow Grove, PA 19090, USA. Electronic address: Kevin.Legg@cfsre.org.

PMID: 34311288
DOI: 10.1016/j.forsciint.2021.110908

Abstract

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.

Keywords: Developmental validation; Forensic biology; Forensic serology; Mass spectrometry; Proteomics.