Assessing protein sequencing in human single hair shafts of decreasing lengths

Abstract

Hair evidence is commonly found at crime scenes and is first analyzed using microscopy techniques. Hair can be processed for DNA analysis, but nuclear DNA analysis may result in a partial or no profile, and mitochondrial DNA analysis is less discriminatory. Single amino acid polymorphisms (SAPs) in hair shaft keratin proteins that result from non-synonymous single nucleotide polymorphisms (nsSNPs) in the genome are being studied as a method of supplementing microscopic comparison of questioned and known hair evidence. Most studies, however, use large amounts of hair (on the order of hundreds of centimeters of hair shaft length), not representative of operational practice in typical forensic casework analyses. Using a recently developed method of hair shaft protein extraction, this study determines how decreasing hair shaft sample length (i.e., 2 cm to 0.12 cm) affects the identification of hair proteins. For example, in 2 cm hair shaft samples, 16 hair shaft keratin proteins, KRT31-40 and KRT80-86, were high-abundant proteins identified with ˜65% average sequence coverage and 44 peptides on average per protein. When the hair shaft samples were decreased to 0.12 cm, this method still identified 15 hair shaft keratin proteins (i.e., except for KRT40) with ˜47% average sequence coverage and 26 peptides on average per protein. This study demonstrates that even with samples as small as 0.12 cm, hair shaft keratin proteins can still be reliably identified and potentially used forensically. Additionally, using the protein extraction technique described in this study, the adequate hair shaft length required for analysis should be in the range of 0.5 cm to 2 cm. Thus, peptide sequencing for SAP identification can be compatible with forensic casework sample sizes.