Development of a Protein‐based Human Identification Capability from a Single Hair

Abstract

Shed human hair (lacking root nuclear DNA) frequently contributes important information to forensic investigations involving human identification. Detection of genetic variation observed in amino acid sequences of hair proteins provides a new suite of identity markers that augment microscopic hair analysis and mitochondrial DNA sequencing. In this study, a new method that completely dissolves single hairs using a combination of heat, ultrasonication, and surfactants was developed. Dissolved proteins were digested and genetically variant peptide (GVP) profiles were obtained for single hairs (25 mm) via high‐resolution nanoflow liquid chromatography‐based mass spectrometry and a novel exome‐driven bioinformatic approach. Overall, 6519 unique peptides were identified and a total of 57 GVPs were confirmed. Random match probabilities ranged between 2.6 × 10−2 and 6.0 × 10−9. The new bioinformatic strategy and ability to analyze GVPs in forensically relevant samples sizes demonstrate applicability of this approach to distinguish individuals in forensic contexts.

Sensitive Method for the Confident Identification of Genetically Variant Peptides in Human Hair Keratin

“…These sequences along with corresponding nonvariant sequences are listed in Appendix S3. Table shows the specific GVP identification for the three methods with three replicate runs for each method, namely our direct method, the modified NaOH + SDS method , and the cleavable surfactant method . For both the direct method and modified NaOH + SDS method, GVP panel results from different fractions are combined in Table .…”

“…The publication by Parker et al showed that these peptides might serve as a source of evidence in addition to DNA for human identification due to several advantages that a hair sample carries: (i) commonly found—on average, humans shed 50–150 hairs per day; (ii) stable—proteins in a hair sample usually last longer and are more resistant to degradation than DNA; and (iii) when good quality DNA is not available, hair proteins may serve as alternative evidence by detecting those GVPs in hair cuticular keratins and other hair proteins. A recent publication by Mason et al described protein‐based or GVP‐based human identification from a single hair as short as 1 inch long. Another recent publication by Carlson et al described a sensitive method to extract proteins from 1 millimeter or less in total length of human anagen head hairs and compared the proteins identified from hair shaft and hair root.…”

“…KEYWORDS: forensic science, genetically variant peptide, hair protein extraction, cuticular keratins, peptide mass spectral library, trace detection In recent publications from Lawrence Livermore National Laboratory (LLNL), genetically variant peptides (GVPs) derived from human hair have been shown to have forensic value (1,2). The publication (1) by Parker et al showed that these peptides might serve as a source of evidence in addition to DNA for human identification due to several advantages that a hair sample carries: (i) commonly found-on average, humans shed 50-150 hairs per day; (ii) stable-proteins in a hair sample usually last longer and are more resistant to degradation than DNA; and (iii) when good quality DNA is not available, hair proteins may serve as alternative evidence by detecting those GVPs in hair cuticular keratins and other hair proteins.…”

“…Hair cuticular keratins have been classified as type I (31‐38) and type II (81‐86) based on the finding that type I keratins are acidic and type II keratins are neutral or basic proteins . Two recent publications from LLNL have collectively identified a total of 88 GVP sites from multiple donors with bulk of hair samples: 32 sites from hair cuticular keratins, 7 sites from cytoskeletal keratins, 22 sites from keratin‐associated proteins, and 27 sites from nonkeratins.…”

“…In this manuscript, we describe a direct protein extraction method (referred as the direct method) that can efficiently extract hair proteins from a single hair shaft less than 1 cm in length. We performed GVP panel analyses and examined experimentally introduced artifactual modifications among three methods: our newly developed direct method and two of previously published methods—NaOH‐based SDS repeated extraction method (we modified it to make it fit in small sample analysis, referred as modified NaOH + SDS method) and ProteaseMAX‐based method (referred as cleavable surfactant method) . Considering the direct method and modified NaOH + SDS method both utilize protein gel electrophoresis to separate extracted proteins, we made further comparisons between these two in‐gel methods for sensitivity and reproducibility.…”