Animal Species and Blood Identification with Peptide Mass Fingerprinting

As more families are acquiring pets and the opportunities for wild animals to appear in human neighborhoods are increasing, the number of cases and accidents involving animals is increasing. Hence, the need to identify animal species from blood left over at accident sites is increasing. Human hemoglobin is used as a marker for human blood. Although tandem mass spectrometry is the dominant methodology used in proteomics research, peptide mass fingerprinting, given its instant applicability, may be useful for screening animal species, as the amino acid sequences of hemoglobin from various animals differ. In this study, solutions that were easily purified─using hemoglobin reagents from humans, Japanese macaques, bears, cattle, goats, sheep, sika deer, pigs, wild boars, dogs, cats, and nutrias─were digested by trypsin, and subjected to database searched using Mascot. No candidate proteins were found in the blood of goats, sheep, sika deer, wild boars, pigs, or nutrias. However, bloodstains from all animal species except nutria (which is not registered in the database) yielded candidates, which were identified as the hemoglobin of origin or its relatives. This difference may be attributed to more contaminants being included in blood. Further narrowing was possible using the average mass obtained via infusion electrospray ionization mass spectrometry measurement of the undigested solution in Mascot results. Saliva, urine, semen, and sweat collected from humans were also examined and searched for mascots, but no hits were obtained. In conclusion, this method may be useful for estimating animal species and identifying blood in forensic science.