Proteomic genotyping offers considerable improvement for the use of hair evidence for individual identification. One advantage is the supplementation of subjective microscopic findings with quantitative values with statistical estimates and experimentally determined error rates. This project aims to dramatically improve the value of hair shafts as crime scene evidence. Genetically variant peptides (GVPs) that are the consequence of non-synonymous single nucleotide polymorphisms (SNPs) that are translated as single amino acid polymorphisms (SAPs). These SNPs are common, well documented and characterized in genetic databases. In aggregate, the resulting random match probabilities can be highly discriminating. The researcher s experiments to optimize the proteomic genotyping approach to individual identification. The specific aims are to (1) maximize the yield of genetically variant peptides from 20 mm of hair for individualization, (2) increase the selection of known genetically variant hair peptides while creating a calibration curve for ancestral classification, and (3) compare instrumental methods to increase the sensitivity of the analysis. In addition to facilitating personal identification, chemical and instrumental optimization is anticipated to permit matching hair sources using their biogeographical backgrounds. The experiments will generate mass spectral data from tryptic digests of African and European hair protein and utilize peptide-identifying software to identify candidate genetically variant peptides. Such peptides will then be confirmed by matching them to non-synonymous single nucleotide polymorphisms in donor genomic DNA. The researchers initial study using optimized temperatures and incubation times for protein reduction and digestion produced random match probabilities of up to 1 in 393 million with 20 mm (~100ug) of hair. Data also indicate that likelihood ratios may be used to differentiate between European and African populations and that using targeted methods of proteomic analysis may increase sensitivity from 20% to 50%. Specifically, targeted methods of proteomic analysis such as Parallel Reaction Monitoring (PRM), Data Independent Acquisition (DIA) and Tandem Mass Tag (TMT) labeling of peptides has been shown by our data and others to be of significant value in increasing the detection limit and sensitivity of endogenous peptides of low abundance. After comparing one targeted method of data acquisition among six subjects, the percentage of true positives increased from 13% to 33%. Current work addresses the establishment of equivalent proteomes between European and African samples in terms of protein expression levels and GVP occurrences. Obtaining genotypes for GVP-associated SNPs from genomic DNA must also be completed. Since GVPs from less abundant proteins are more likely to be observed with increased efficiency of hair digestion and instrumental detection, this work will increase the number of GVP identifications and also increase statistical discrimination.
Note: This project contains a research and/or development component, as defined in applicable law," and complies with Part 200 Uniform Requirements - 2 CFR 200.210(a)(14).