Estimating animal species from blood using in-gel digestion peptide mass fingerprinting

Increasing numbers of domestic and wild animals in residential environments have contributed to a rise in animal-related incidents. As a result, the forensic identification of animal species from trace blood evidence has become increasingly important. In our previously reported simplified protocol for hemoglobin peptide mass fingerprinting (PMF), applicability was limited to mammals and required comparatively large blood volumes. To address these constraints, we implemented an in-gel digestion PMF workflow and sought to determine the optimal protein content for analysis. Pooled blood from 10 animals was subjected to SDS-PAGE, followed by Mascot-based protein identification. Subsequently, to evaluate species applicability and the suitability of the method for bloodstain analysis, in-gel digestion PMF was performed on blood samples from 21 species and bloodstains from 10 species at the optimized protein load. In nearly all specimens, hemoglobin derived from the target or phylogenetically related species yielded the highest protein scores. Overall, in-gel digestion PMF enables species estimation across a broad range of forensic animal samples while requiring a small blood volume. Given that PMF can be conducted using a relatively low-cost single mass spectrometer, this approach has the potential to advance the adoption of forensic proteomics.