Optimal Processing for Proteomic Genotyping of Single Human Hairs
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ABSTRACT: The human hair proteome offers new insight in the field of human identification. Hair often contains DNA that has been degraded due to the process of cornification. The smaller fragments of DNA present a challenge for obtaining useful information concerning the identity of the donor. Proteomic genotyping offers an alternative approach which starts with protein, a biomolecule abundant in the hair shaft. Nonsynonymous single nucleotide polymorphisms may be detected in the sequence of these proteins, allowing the inference of SNP genotypes. Population genetics-based information from these genotypes may be used to calculate random match probability or even infer ancestry. The current challenge of this research is to optimize processing chemistry in order to maximize genetic information from a single human hair shaft. Results indicate that optimal conditions for proteomic analysis of a single human hair include 6 hrs of reduction with 100 mM dithiothreitol at room temperature, alkylation with 200 mM iodoacetamide for 45 min, and 6 hrs of digestion with two 1:50 (enzyme:protein) additions of stabilized trypsin at room temperature, with stirring incorporated into all three steps. Our final conditions using optimized temperatures and incubation times for disulfide reduction and protein digestion produced random match probabilities of up to 1 in 624 million from a single hair with a median value of 1 in 1.1 million, compared to a maximum random match probability of 1 in 1380 and a median value of 1 in 24 for the original processing method.
INSTRUMENT(S): Q Exactive
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Hair Shaft
SUBMITTER: Glendon Parker
LAB HEAD: Glendon John Parker
PROVIDER: PXD016155 | Pride | 2020-06-10
REPOSITORIES: Pride
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