Determination of the Time since Deposition of Blood Traces Utilizing a Liquid Chromatography–Mass Spectrometry-Based Proteomics Approach

Knowledge about when a bloodstain was deposited at a crime scene can be of critical value in forensic investigation. A donor of a genetically identified bloodstain could be linked to a suspected time frame and the crime scene itself. Determination of the time since deposition (TsD) has been extensively studied before but has yet to reach maturity. We therefore conducted a proof-of-principle study to study time- and storage-dependent changes of the proteomes of dried blood stains. A bottom-up proteomics approach was employed, and high-resolution liquid-chromatography–mass-spectrometry (HR-LC–MS) and data-independent acquisition (DIA) were used to analyze samples aged over a 2 month period and two different storage conditions. In multivariate analysis, samples showed distinct clustering according to their TsD in both principal component analysis (PCA) and in partial least square discriminant analysis (PLS DA). The storage condition alters sample aging and yields different separation-driving peptides in hierarchical clustering and in TsD marker peptide selection. Certain peptides and amino acid modifications were identified and further assessed for their applicability in assessing passed TsD. A prediction model based on data resampling (Jackknife) was applied, and prediction values for selected peptide ratios were created. Depending on storage conditions and actual sample age, mean prediction performances ranges in between 70 and 130% for the majority of peptides and time points. This places this study as a first in investigating LC–MS based bottom-up proteomics approaches for TsD determination.