Enhanced Genetically Variant Peptide Profiling from Human Hair Using Multiple Enzymes

Genetically variant peptides (GVPs) derived from human hair proteins have been studied as evidence for forensic human identification. For this purpose, trypsin is generally routinely used as the protease for peptide generation. We hypothesized that the use of different proteases can expand sequence coverage in the hair proteome and, therefore, increase the chance of a variant peptide being identified and confirm the findings from tryptic digests. Since keratins and keratin-associated proteins are the main components of the hair proteome, we examined several alternative enzymes other than trypsin, including keratinase, chymotrypsin, and Glu-C. We found that the use of multiple enzymes led to enhanced GVP profiling and, therefore, an increase in the confidence of GVP identification.