Date of Award

2021

Document Type

Dissertation

Degree Name

Ph.D.

Department

Biological Sciences

First Advisor

Phillip B. Danielson

Second Advisor

Jim Fogleman

Third Advisor

Nancy Lorenzon

Fourth Advisor

Scott Horowitz

Fifth Advisor

Keith Miller

Keywords

Forensic science, Mass spectrometry, Proteomics, Serology

Abstract

Protein mass spectrometry (MS) has emerged as a technique to supplant traditional serological tests for body fluid identification. It was hypothesized that proteomic techniques would surpass the sensitivity and specificity of traditional serological techniques. An automated workflow coupled with protein MS has been developed for the confirmatory identification of five biological fluids. A developmental validation was completed, assessing parameters such as reproducibility, sensitivity, ion suppression, and limit of detection. Implementation was determined through tandem sample processing by MS, traditional serological tests, and standard DNA profiling methods. The MS approach offered superior detection limits while also providing true confirmatory results, producing an unambiguous identification of body fluids to the point where the technology can be considered comparable to DNA profiling.

An extensive study was conducted to evaluate the effects of personal lubricants on biomarker detection in sexual assault evidence. Lubricants have the potential to inhibit protease activity, displace hydrophobic markers during solid phase extraction, and suppress ion detection during MS analysis. Three studies were performed: (1) determination of vaginal fluid biomarker detection from vaginal swabs fortified with lubricant; (2) the effect of lubricant formula on seminal fluid and saliva biomarker detection was established; and (3) the detection of biomarkers condoms. Data was interpreted by the overall peak area response (PAR) of the target biomarker, biomarker PAR in relation to internal standard, and PAR of digestion control protein.

Multi-stage workflows associated with proteomic analysis remain a major hurdle towards the adoption of the technique in caseworking laboratories. A streamlined sample-to-results workflow has been developed using peptidomic analysis, allowing for straightforward preparation versus bottom up methodologies. Low molecular weight proteins were extracted and data was acquired using an orbitrap-quadrupole HRMS. Numerous protein biomarkers have been characterized in human seminal fluid, saliva, and vaginal fluid.

In conclusion, the implementation of the protein MS approach offers an advantageous relationship between a positive identification and downstream DNA testing, including the capacity to deliver confirmatory contextual information in a criminal investigation. Furthermore, lubricant type does affect the ability to accurate identify protein biomarkers. And lastly, the research presented will demonstrate the use of peptidomic analysis for the confirmatory identification of biological fluids in SA type evidence.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Provenance

Received from ProQuest

Rights holder

Catherine O'Sullivan Brown

File size

440 pgs

File format

application/pdf

Language

en

Discipline

Biochemistry, Analytical chemistry, Biology

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