Author: Sathisha Kamanna
Kamanna, Sathisha, 2018 Mass Spectrometry-based Proteomic Analysis of Forensic Body Fluids, Fingermarks and Fingernail Traces, Flinders University, College of Science and Engineering
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In the past decade, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and mass spectrometry imaging (MSI) have been widely used in clinical and/or biological sciences for the detection, differentiation and mapping of differentially expressed proteins or peptide markers within tissue sections. Recently, MALDI-MS techniques have been applied to forensic science for the analysis of organic small molecules (e.g., illicit drugs and metabolites) and protein biomarkers for identification of body fluids such as blood, saliva, seminal fluid, urine, vaginal fluid and sweat in fingermarks.
The most important forensic test, DNA profiling, can indicate from ‘whom’ biological traces originated. However, it is also very important to determine the ‘type’ of body fluid and differentiate it from non-human fluids, thus providing evidence as to the circumstances surrounding the crime. Furthermore, biological substances such as blood or vaginal fluid and other body fluid mixtures may become smeared on the fingers of the victim or suspect or trapped under their fingernails. If either person touches an object with their “contaminated” finger then a “contaminated” fingermark may be deposited. Such marks could be great value as they can identify not only who deposited the mark, but also who they touched and which part of the body they touched. These trapped biological traces are extremely valuable information to forensic investigation. There is a lack of confirmatory tests for many body fluids and current available presumptive tests often produce false positives, suffer from interferences, lack human-specificity, and cannot distinguish between menstrual blood and venous blood, menstrual fluid and vaginal fluid or mixtures of other bodily fluids.
In this study, mass spectrometry (MS)-based in-situ “top-down” and “bottom-up” proteomics analysis was carried out for the direct identification of “protein markers” in body fluids and their mixtures. The work describes a streamlined MALDI-ToF MSbased proteomic methods for the identification of body fluid traces recovered from different, important substrates (e.g., various types of fabric fibres, cotton swabs and nylon microswabs), directly in situ on these substrates, and from enhanced latent fingermarks (including “lifted” enhanced marks). Direct identification of intact proteins (“top-down” analysis) on fabric and microswab fibres using MALDI-MS is often challenging due to low mass axis resolution and accuracy, especially in mixture of body fluids. Thus, the use of “bottom-up” proteomics (peptide mass fingerprint analysis) and in-situ MS/MS (tandem mass spectrometry) analysis followed by de novo sequencing was investigated in order to enhance the specificity and reliability of protein biomarker identification. The described mass spectrometry methods for the direct identification of differentially expressed protein “markers” in native or dilute solutions of body fluids and in aqueous suspensions of dried body fluid particles on different substrates, offers significant advantages compared to other classical proteomics, including speed of analysis, reduced the cost of complex requirements and elimination of crosscontamination. Even though body fluids of relevance to forensic science are complex mixtures of proteins, they each contain characteristic proteins that are abundantly expressed and they ionize in preference to other proteins that are less abundant. Although MALDI-ToF techniques do not have the performance to discover trace biomarkers in the wide range of tissues and fluids relevant to medical science, the techniques appear to be fit for the purpose of identifying the small range of body fluids that are encountered in forensic biology.
MALDI-MSI techniques were applied for the direct identification and mapping of foreign body fluids (such as blood, vaginal fluid) in fingermarks, including latent fingermarks that were enhanced using silver/black magnetic powder or Amido Black and lifted using double sided Kapton adhesive tape. MALDI-TOF MS provided greater species–specific information as compared to the popular immunochromatographic screening test for human blood (Hematrace® ABAcard®) and fingermark protein staining methods (Amido Black). This thesis also describes for the first time “top-down” and “bottom-up” mass spectrometry-based proteomics approaches for the identification and differentiation of haemoglobins on bloodied fingermarks. It was shown that blood from Australian native animals readily can be differentiated with human blood. Even haemoglobins in mixed blood (human and animal blood) was successfully detected and imaged in bloodied fingermarks. Small endogenous molecules were detected in lifted ridge patterns.
Differential proteome analysis using nLC-ESI-qTOF MS/MS showed a high confidence (>95%) identification and confirmation of body fluid protein markers and detection of unknown haemoglobins from Australian marsupials blood samples.
A schematic work flow presented in this thesis describes a complementary forensic ‘proteo-genomic’ analysis on a single micro-swab for the direct identification of the “type” of biological fluid present under fingernails and the identity of its “donor”. The analytical strategy (forensic “toolbox”) demonstrated in this work involves two complementary techniques, direct PCR DNA profiling and mass spectrometry-based protein biomarker detection. Post-direct PCR solutions can be used successfully for proteomic identification of haemoglobins, which provides both DNA profiles and proteomic information from the same sample.
The applicability of novel MS-based proteomics (“top-down and “bottom-up”) and tandem mass spectrometry analysis followed by de novo sequencing and MALDI-MSI techniques presented in this thesis and the published articles offer significant potential for forensic biology applications.
Keywords: Forensic, proteomics, mass spectrometry, body fluids
Subject: Forensic & Analytical Chemistry thesis
Thesis type: Doctor of Philosophy
Completed: 2018
School: College of Science and Engineering
Supervisor: Paul Kirkbride