Lions and Tigers and Bears Oh My: Rapid Identification of Animal Species Blood Using Paper Spray Mass Spectrometry

Violence against animals, including poaching and illegal hunting, can lead to complex crime scenes where it is difficult to identify the animal species involved, particularly when the evidence is mutilated or only residual blood remains. Current approaches such as DNA analysis, involve expensive and time-consuming processes that often slow down investigations. Paper spray mass spectrometry (PS-MS) is an inexpensive way to rapidly detect drugs, proteins, and metabolites in biological samples. Reported here are the results of efforts to develop this method as a rapid and inexpensive tool for the identification of animal species from residual blood. It is based on the presence of hemoglobin in the blood of most animals, which has unique alpha and beta chains based on species-specific differences in their primary protein sequences. Using PS-MS, these species-specific alpha and beta chain masses can be detected and used to identify the species. Accordingly, whole blood samples of multiple individuals from seven mammalian species, Bos taurus (domestic cow), Capra hircus (domestic goat), Cervus elaphus (red deer), Equus caballus (horse), Ovis aries (sheep), Sus Scrofa (wild hog) and Homo sapiens (human), were analyzed using PS-MS, and the species-specific alpha and beta chains were identified. The alpha and beta chain masses were compared to previously reported results and the uniport database to confirm species identities. PS-MS enabled successful species identification with the findings matching published results. For example, the domestic cow sample matched the alpha and beta chain literature values at 15053 m/z and 15954 m/z respectively. Similarly, in alignment with published reports for domestic goats, the values for the alpha and beta chains exhibited intraspecies variations with masses corresponding to reported m/z ranges. These results show that PS-MS is a less expensive and faster method to reliably identify animal species from residual blood.