Alternative LCMS/MS Platforms and Data Acquisition Strategies for Proteomic Genotyping of Human Hair Shafts (original) (raw)
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Assessing Protein Sequencing in Human Single Hair Shafts of Decreasing Lengths
Forensic Science International: Genetics
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 High-Yield Two-Hour Protocol for Extraction of Human Hair Shaft Proteins
Proteome analysis of the human hair remains challenging due to the poor solubility of hair proteins and the difficulty in their extraction. In the present study, we have developed a rapid extraction protocol for hair shaft protein using alkaline-based buffer. The new protocol accelerated the procedure by reducing the extraction time from at least a day to less than two hours and showed a protein recovery of 47.3 ± 3.72%. Further analyses of the extracted protein sample through sodium dodecyl sulfate polyacrylamide gel electrophoresis and Quadrupole-time-of-flight mass spectrometry analysis unveiled a total of 60 proteins, including 25 that were not previously reported. Identification of these proteins is anticipated to be crucial in helping to understand the molecular basis of hair for potential applications in the future.
Forensic Science International: Genetics
The microanatomy of human hair differs as a function of the site of origin on the body. This was a major consideration when anatomical features of hair were used as a means of comparison and human identification. Recent advances have demonstrated that proteomics of the hair shaft can be used to develop profiles of protein abundance and genetically variant peptides, the latter in turn being used to infer genotypes of SNP alleles. Because the profile of proteins would be expected to change as hair anatomy changes, it is an open question if the profile of genetically variant peptides will also change. While some sample to sample variation is expected, a potential drawback of using genetically variant peptides to infer an individual genotype is that the proteomic profile might change as a function of body site origin as well as an individual's genotype. The hypothesis in this study is that the profile of hair shaft genetically variant peptides depends more on an individual's genotype than on the site of hair shaft origin. To test this an analysis of both protein expression levels and genetically variant peptides was conducted on 4 body sites (scalp, axillary, beard and pubic hair) from 5 individuals with 4 biological replicates. Levels of protein expression were estimated using label-free quantification on resulting proteomic mass spectrometry datasets. The same datasets were then also analyzed for the presence of genetically variant peptides. This study demonstrates that the protein profiles of hair shafts varied as a function of somatic origin. By contrast the profile of genetically variant peptides, and resulting inferred genotype of SNP alleles, were more dependent on the individual. In this study random match probabilities ranged up to 1 in 196. Individual identification based on genetically variant peptides therefore can be obtained from human hair without regard to the site of origin. If the site of hair shaft origin was legally relevant then microscopic analysis is still necessary. This study demonstrates the utility of proteomic analysis for extracting forensic information from hair shaft evidence.