Investigating the peptide profile of decomposition fluid by liquid chromatography mass spectrometry to assist in the determination of the post-mortem interval (PMI)

An accurate estimation of post-mortem interval (PMI), or time since death of an alleged victim, is of utmost importance in criminal investigations. Several biochemical approaches have been attempted but a definitive method for time since death estimations has yet to be identified. Recent studies have demonstrated that protein biomarkers are a promising alternative to previously investigated biochemical products, but the use of older analytical methodologies, together with a reliance on specific tissues, has limited the analysis and interpretation of the data. This thesis describes investigations utilising more sophisticated proteomic techniques to evaluate the rate of protein degradation in decomposition fluid. The primary aim of this research was to characterise the peptide profile of decomposition fluid generated under a variety of experimental (laboratory-based) and field-based environmental conditions (summer versus winter), and to evaluate its potential for PMI estimation. Domestic pig (Sus scrofa domesticus) cadavers were used to model the human decomposition process. The peptide components of decomposition fluid were analysed using high performance liquid chromatography-time of flight mass spectrometry, followed by high performance liquid chromatography-triple quadrupole mass spectrometry. Observations of physical characteristics were also recorded in order to determine a total body score for each cadaver. Fluid analyses revealed that a range of protein-specific peptides (originating from haemoglobin subunit alpha and beta, beta-enolase, creatine kinase and lactate dehydrogenase) were consistently generated during the decomposition process, regardless of temperature or trial conditions. These peptides, when averaged on each sampling day, were identified more than 50% of the time during the course of each decomposition trial. Furthermore, when mapped against reference sequences, degradation patterns for haemoglobin subunits alpha and beta were similar when expressed in accumulated degree days (ADD) and when adjusted for differences in temperature, regardless of trial conditions. Principal component analysis identified 29 peptides, originating from haemoglobin subunits alpha and beta, creatine kinase, beta-enolase and lactate dehydrogenase, that contributed to differences in the peptide profile of samples collected during the early period (days 6–12 and day 2 in winter and summer, respectively) and later period (days 24–34 and days 8–10 in winter and summer, respectively) of the decomposition process. Fold changes in the spectral profile for eight of these peptides were found to be significantly different between these periods. Trends displayed by six of the eight peptides, three of which were derived from haemoglobin subunit beta, one from beta-enolase and two from lactate dehydrogenase, indicated that a factor other than temperature impacted the rate of protein degradation. In contrast, the trends exhibited by the remaining two peptides derived from haemoglobin subunit alpha and creatine kinase, suggested that temperature was the major external factor involved in their production. Further analyses identified several peptides (one originating from haemoglobin subunit alpha, three from haemoglobin subunit beta and three from lactate dehydrogenase), the rate of production of which, could be correlated with total body score and with the early stages of decomposition. Any biochemical marker employed to help define the post-mortem interval will be subject to inherent variability. However, the results described in this thesis demonstrate that peptide analysis during the decomposition process is a promising approach, which, with further development may prove more reliable than previously studied biochemical parameters