Bottom-up in situ proteomic differentiation of human and non-human haemoglobins for forensic purposes by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry

RationaleThe detection and identification of human blood on crime-related items are of particular relevance to many investigations because shed blood can provide evidence of violent contact between individuals. However, for any detection and identification technique, specificity is a critical performance characteristic to assess; that is, whether the technique has the capability to differentiate between human blood (which usually is of relevance to a criminal investigation) and non-human blood (which usually would not be associated with a crime but may be detected incidentally). MethodsMatrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) approaches using top-down (detection of intact proteins) and bottom-up (detection of tryptic peptide markers) were used to detect and identify haemoglobin in blood from humans and from a range of Australian native mammals; the technique could be carried out directly on blood stains without the need to extract proteins (i.e., in situ measurement). Imaging of haemoglobin was achieved in bloodied fingermarks, including those that had been enhanced using two industry standard fingermark enhancement processes. ResultsDifferentiation of intact haemoglobin proteins in human and non-human blood using top-down MALDI-TOF-MS was difficult. However, in situ bottom-up approaches using tandem mass spectrometry (MS/MS) and de novo sequencing of tryptic digest peptides allowed unambiguous differentiation. Imaging mass spectrometry of human haemoglobin, even when it was mixed with animal blood, was achieved in bloodied fingermarks that had been enhanced using two common processes (staining with Amido Black or dusted with magnetic powder) and lifted using adhesive tape. ConclusionsThe MALDI-TOF-MS-based in situ bottom-up proteomic methodology described here shows great promise for the detection of human blood and even imaging of blood in bloodied fingermarks. The approach is sensitive, can differentiate between human blood and that from many animals (including several Australian native animals), and can be implemented after traditional crime scene fingermark enhancement techniques have been carried out.