Author Identifier
Date of Award
2026
Keywords
forensic proteomics, genetically variant peptides, mass spectrometry, proteomic genotyping, single nucleotide polymorphisms
Document Type
Thesis
Publisher
Edith Cowan University
Degree Name
Doctor of Philosophy
School
School of Science
First Supervisor
Hayley Abbiss 
0000-0002-6821-5564
Second Supervisor
Joel Gummer 0000-0002-3609-3398
Third Supervisor
Glendon Parker
Fourth Supervisor
Armaghan Shafaei Darestani 0000-0002-5763-531X
Abstract
Introduction: In the absence of DNA, proteomic genotyping is a proposed means of associating biological forensic evidence with an individual. Complementary to DNA-based methods, this technique has the potential to assist in situations where DNA is poorly recovered or absent. Genetically variant peptides (GVPs) within the hair shaft can be used to infer corresponding SNP genotypes. Hair evidence is forensically significant due to being chemically stable and abundantly shed in the environment.
This research aimed to develop a robust forensic workflow for the isolation, analysis, and bioinformatic interpretation of GVPs within the human hair shaft from the Western Australian population. Method enhancements were considered such that they would be viable for translation from a research and development phase to a forensic laboratory environment in the future.
Methods: For sample preparation, sonication followed by bead mill homogenisation was compared against the use of a stir flea for sample homogenisation. Suspension trapping (S-Trap) was also compared against liquid-liquid extraction (LLE) based methods as an alternative means of sample clean-up. The effects of trap column use, column length, injection volume, and carry-over were additionally assessed by nano LC-MS/MS analysis via data dependant acquisition (DDA). The S-Trap method was further investigated to determine the sensitivity and reproducibility of detected true positive GVPs.
Processed data from a recruited cohort of 160 individuals was interrogated for the development of individual selection criteria required for identification of true positive peptides within a 474 GVP panel. Genetic validation of the proteomic results produced a peptide validation matrix. False positive peptide assignments identified from the matrix were further investigated to determine whether they could be distinguished between corresponding true positive peptide matches.
Population-specific data from the 160 sample set was further examined on the basis of hair proteome profiles, GVP profile, and average abundance of peptides detected across a broad variety of samples. Proteomic results between bleached and unbleached hairs, and between varying age groups, were compared. Candidate novel GVPs specific to the recruited population were searched amongst the data set to determine any that could be added to the wider GVP panel.
Results: An enhanced suspension trapping (S-Trap) method was selected as the final preparative approach, providing reproducible results and a greater number of true positive GVPs detected compared to the baseline method from literature used for comparison. The use of a trap column and a longer acquisition method additionally resulted in a greater number of true positive peptide detections compared to a direct injection approach and use of a short separation column. A reduction in injection volume combined with the implementation of a wash method between sample injections was found to reduce the incidence of carryover.
Interrogation of processed data from the 160 sample set allowed for development of peptide-specific selection criteria, necessary for GVPs within the working panel to be confirmed reliably. Abundance cutoffs were established for true positive peptides that could be differentiated from false positive matches. False positive assignments that could not be differentiated were removed from the GVP panel to produce a refined working GVP list.
A final population study of the 160 sample set produced population-relevant GVP profiles, consisting of East Asian, European, South Asian, Kurdish, Pacific Islander, and Admixed individuals. The number of peptides detected and discriminating power were significantly reduced for bleached hairs over unbleached; conversely, there was so significant difference in the numbers of proteins and GVPs between the two groups. There was additionally an absence of significant age-related differences across the number of peptides, GVPs, RMPs, and protein groups detected. Peptide variant discovery also introduced a refined list of 44 novel GVPs specific to the population study.
Conclusions: This work demonstrated the successful application of an enhanced workflow for the purposes of proteomic genotyping. True positive peptide identifications per sample from the developed methods increased compared to baseline methods in literature, adding discriminatory power. The greatest advancement towards translating the methodology to a forensic laboratory setting was through the development of individual peptide selection criteria, allowing robust identification of true positive peptides and rapid resolution of many false positive peptide matches. Ultimately, this work led to the development of a refined GVP panel of sensitive peptides that can be reliably identified as true positive matches within experimental data.
Access Note
Access to this thesis is embargoed until 9th October 2031
Recommended Citation
Tidy, R. J. (2026). Development of a routine forensic process for individual identification using genetically variant peptides in human hair evidence. Edith Cowan University. https://doi.org/10.25958/geyv-5w19