Assessing Single-Source Reproducibility of Human Head Hair Peptide Profiling from Different Regions of the Scalp.

Neither microscopical hair comparisons nor mitochondrial DNA sequencing alone, or together, constitutes a basis for personal identification. Due to these limitations, a complementary technique to compare questioned and known hair shafts was investigated. Recently, scientists from Lawrence Livermore National Laboratory's Forensic Science Center and other collaborators developed a peptide profiling technique, which can infer non-synonymous single nucleotide polymorphisms (SNPs) preserved in hair shaft proteins as single amino acid polymorphisms (SAPs). In this study, peptide profiling was evaluated to determine if it can meet forensic expectations when samples are in limited quantities with the possibility that hair samples collected from different areas of a single donor's scalp (i.e., single source) might not exhibit the same SAP profile. The average dissimilarity, percent differences in SAP profiles within each source, ranged from 0% difference to 29%. This pilot study suggests that more work is needed before peptide profiling of hair can be considered for forensic comparisons.

Assessing protein sequencing in human single hair shafts of decreasing lengths.

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.

A study of the intrinsic variability and the effect of environmental conditions on the formation of a postmortem root band.

A postmortem root band (PMRB) is defined as "an opaque ellipsoidal band composed of a collection of parallel elongated air/gas spaces and is approximately 0.5mm above the root bulb and about 2mm below the skin surface" [1]. It is generally accepted that it can appear in the root of hairs attached to remains during decomposition [1]. This study aimed to investigate the underlying cause and mechanism of PMRB formation. This was done (i) by observing the overall frequency and the intrinsic variability in anagen hairs containing a PMRB collected across five regions of a human decedent's scalp at three time points, and (ii) by determining if PMRB-like features can be induced via immersion in in-vitro controlled environments of anagen hairs plucked from the scalp of a human decedent (ex-situ postmortem hairs) not containing a PMRB. The results of the first objective illustrated that as time since death increased, the frequency of hairs containing a PMRB across the scalp sampling regions increased and the intrinsic variability decreased. The results of the second objective demonstrated that both an aqueous environment and microbial activity are essential for the formation of PMRB-like features. This study was the first to statistically analyze the intrinsic variability of PMRB formation, as well as the first to induce PMRB-like features in roots of ex-situ postmortem hairs.

Protein extraction from human anagen head hairs 1-millimeter or less in total length.

A simple method for extracting protein from human anagen (i.e., actively growing hair stage) head hairs was developed in this study for cases of limited sample availability and/or studies of specific micro-features within a hair. The distinct feature segments of the hair from one donor were divided lengthwise (i.e., each of ∼200-400 µm) and then pooled for three individual hairs to form a total of eight composite hair samples (i.e., each of ∼1 mm or less in total length). The proteins were extracted, digested using trypsin, and characterized via nano-flow liquid chromatography tandem-mass spectrometry (nLC-MS/MS). A total of 63 proteins were identified from all eight protein samples analyzed of which 60% were keratin and keratin-associated proteins. The major hair keratins identified are consistent with previous studies using fluorescence in situ hybridization and nLC-MS/MS while requiring over 400-8000-fold less sample. The protein extraction method from micro-sized human head hairs described in this study will enable proteomic analysis of biological evidence for cases of limited sample availability and will complement hair research. For example, research seeking to develop alternative non-DNA based techniques for comparing questioned to known hairs, and understanding the biochemistry of hair decomposition.