Application & Background

Detection, confirmation and visualisation of blood at crime scenes informs on the dynamics of the bloodshed and on the nature of the crime. It may be crucial to also indicate foul play in a murder without a body. Current presumptive methods used by crime scene investigators are prone to false positives and hence a reliable confirmatory test is paramount. MALDI MS Profiling and Imaging (MALDI MSP/I) have proven to be suitable confirmatory tests to detect and map the presence of blood and its origin (human or animal)1 in fingermarks and reconstruct the ridge pattern for suspect identification.2-3 Specifically, a method has been developed combining the use of bottom-up proteomics and MALDI MSP/I to detect and visualize blood through blood specific proteins and in particular from the peptides generated by the in situ enzymatic digestion.

In this application, trypsin is sprayed at a low flow rate across the blood mark using the HTX M3+ Sprayer. A lower flow rate allows to minimise enzyme consumption. Following trypsin application and incubation, the sample is returned to the sprayer for matrix application. Matrix application is performed at a higher flow rate, but with the same pump used for enzyme spraying, without needing any manual modifications to the instrument. The automated PumpScriptsTM on the HTX M3+ Sprayer (Figure 1) provide thorough and hands-free cleaning of the Sprayer between enzyme and matrix spray cycles, which is essential for optimal application of these two distinct chemicals. An overview of this workflow is shown in Figure 2.

Spray conditions specific for blood fingermark samples are needed for both the enzyme and the matrix application in this workflo due to the blood fingermark being less securely attached to slides compared to tissues. As the ridge pattern and details provide biometric information, spray conditions such as gas pressure, velocity, and track spacing must be optimized to preserve the ridge detail integrity.

Experimental

experimental design & Fingermarks

Human blood fingermarks were prepared on poly-L-lysine coated slides. Human blood was obtained by pricking a clean finger with a single-use lancet and contained in EDTA tubes. Ten microliters of blood were spotted on a slide to wet a silicon fingertip which consequently generated a blood mark by contact with another clean glass slide. The blood mark was fixed in100% methanol for 1 hr before being left to dry.

Enzyme and Matrix Application using the HTX M3+ Sprayer

Using the HTX M3+ Sprayer, trypsin (Table 1) was applied to the slide using the parameters displayed in Table 2. After trypsin spraying, the slides was placed "face up" in a humidity chamber consisting of a silicon sealed tub and containing tissue paper saturated with a 50:50 methanol:water solution. The sample was left to incubate for 3 hrs in a 37°C oven. The slide was then removed from the humidity chamber, and the bottom of the slide was dried using a KimWipe. Using the HTX M3+ Sprayer, α-cyano-4-hydroxycinnamic acid (CHCA) matrix (Table 1) was applied to the slide using the parameters displayed in Table 2.

MALDI MSI

All imaging experiments were performed on a QTOF G2 HDMS Synapt MALDI mass spectrometer (Waters Corporation, Manchester, UK) in positive ion mode using a 1 KHz Nd:YAG laser. Each pixel was collected at a 100 µm spatial resolution.

data collection and analysis

The data were collected and visualized using HD Imaging (HDI) software (Waters Corporation, Manchester, UK). Putative peptide identifications were manually made using an in-house blood peptide database. Blood specific peptide-deriving proteins such as hemopexin, erythrocyte protein band 4.2 (EPB 4.2), serotransferrin, prothrombin, apolipoprotein A1, serotransferrin, ceruloplasmin and complement C3 α were searched;. Images were all normalized to the total ion current (TIC); and to aid visualization of the ridge detail, brightness and contrast were then adjusted by the same level in each image shown. Exclusion mass lists, including matrix/ matrix cluster/ adduct) and trypsin autolysis m/z peaks were generated and used to prevent the assignment of irrelevant m/z signals.

results

Haem and blood specific peptide signatures were imaged in blood fingermarks. Each ion signal m/z can be visualized as heatmaps across the slide, and an overlay is also shown, Figure 3. Quality of fingermarks clarity, continuous ridge flow, and details (minutiae) varied depending on the species imaged with haem providing a grade 4 mark (according to the Home Office grading scheme) (5). While haem is not a result of the trypsin digestion, its visualization enables to appreciate the high quality of matrix application across the fingermark. The presence of hemoglobin alpha and beta peptides at nominal m/z 1275 and 1530 can be used to confirm the human provenance of the blood detected.