# Proteomics in Forensic Analysis: Applications for Human Samples
## 3 Applications of Forensic Proteomics for Human Samples  
### Hair Proteome  
Hair is often available at crime scenes. It is formed by keratinization of epidermal keratinocytes. Hair is physically flexible and robust due to its components, which are primarily coiled-coil proteins with intermolecular disulfide [131]. It generally persists under different environmental conditions. However, hair may not be a favorable sample for DNA analysis because DNA can be extensively degraded due to the keratinization process [132]. A proteomic approach can be performed using hair for individual identification. Notably, mitochondrial genome and proteome profiles have been obtained from hair samples using a combined workflow with high compatibility [133]. A recent study proposed a gel-based proteomic approach to analyze hair samples with high sensitivity, and built a library containing all identified peptides derived from hair [134]. In forensic science, hair can be used in different applications. In a previous study on human hair, [PERSON] et al. used shotgun proteomics (i.e., bottom-up proteomics using LC-MS/MS) to identify ethnic-based differential proteomic signatures among different ethnic groups, including Caucasians, African-Americans, Kenyans, and Koreans [36]. It was found that differences among ethnic groups mainly relied on the levels of keratin-associated proteins in hair (Figure 2a). A recent study demonstrated that keratin peptides of human hair could be used to distinguish gender (using type II-keratin K81, K83, and K86 peptides) and ethnicity (using type I and type II keratin K33b, K81, K83, and K86 peptides) [26]. Another study reported the use of shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects to obtain individualizing and biogeographic information such as ethnicity [18]. Genetically variant peptides (GVPs) containing single amino acid polymorphisms (SAPs) were found in the proteomic datasets and then used to impute the status of non-synonymous single nucleotide polymorphisms (nsSNPs) alleles in subject genomes. It should be noted that mitochondrial DNA and nsSNPs have been used to identify ethnicity, race, and gender [135]. GVPs containing SAPs have been determined by shotgun proteomics for the same purpose because they result from these DNA nsSNPs [136]. In the study by [PERSON] et al., the probability of detecting nsSNP alleles or allele combinations in each gene (Pr(nsSNP gene profile | population)) was calculated and multiplied together to estimate the probability of an overall individual nsSNP profile in the population (Pr(profile | population)) [18]. Thus, European-American and African-American subjects could be differentiated by imputed nsSNP profiles (Figure 2b). In addition, likelihood ratios (LRs) for European relative to African populations were calculated for each individual: LR(EUR/AFR) = Pr(profile | EUR population)/Pr(profile | AFR population). As shown in Figure 2c, the European-American hair shaft protein samples had LRs of \(6.50\times 10^{-1}\) to \(5.85\times 10^{3}\), which were higher than those from African-American and Kenyan samples (\(1.15\times 10^{-3}\) to \(1.07\times 10^{1}\) and \(9.9\times 10^{-3}\) to \(1.21\times 10^{1}\), respectively). The findings in this study suggest great potential of imputed nsSNP allele profiles obtained from hair shatt proteins in determining the biogeographic ancestral background of individuals with quantifiable statistical information. Recently, this group developed and optimized a proteomic workflow to allow individual identification from a single human scalp hair with a length of 2 cm [37]. The optimal conditions were as follows: (1) reduction with 100 mM dithiothreitol at room temperature for 6 h, (2) alkylation with 200 mM iodoacetamide for 45 min, and (3) tryptic digestion (enzyme:protein ratio of 1:50, addition twice at 0 h and 3 h) at room temperature.