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For the past 7 years, Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) based methods have been developed and published for the forensic detection of blood in stains and fingermarks. However, in the view of adoption in an operational context, further investigation into the capabilities and limitations of this approach must be conducted. The refinement and testing of this approach must also be tailored to the requirements of the end users, enabling them to address the specific circumstances most encountered in a forensic scenario. The present study delves deeper into the assessment of the applicability of MALDI MS based strategy for the reliable and robust detection of human blood through: (i) a semi-qualitative assessment of the sensitivity of the method, (ii) a wider investigation of the compatibility of the method with the prior application of commonly used presumptive tests and (iii) assessment of the specificity of the method (when blood is present in mixture with other biofluids) and of its robustness, by assessing blood detection from a range of porous materials. The findings strengthen the evidence supporting the adoption of MALDI MS based approaches as a confirmatory test for the forensic detection of human blood in an operational context.

For decades, the analysis of fingerprints (FP) has been used as the primary biometric mean of human identification. In parallel, the chemical analysis of latent fingermark (LFM) with particular reference to “touch chemistry”, offers additional intelligence to forensic examination; as such, continuous improvements in the versatility and sensitivity of detection of the molecular make up of FM is of obvious importance. In this light, we propose a facile approach to synthesize magnetic carbon nanoparticles (MCNPs) from candle soot for extraction and detection of endogenous and exogenous FM components. Initially, endogenous components of FM including fatty acids (FAs), squalene and triacylgycerols (TAGs), were successfully extracted and detected using the developed MCNPs and surface assisted laser desorption/ionization-mass spectrometry (SALDI-MS). Furthermore, the MCNPs enabled the detection of exogenous substances including antihistamine, β-blocker, antibiotic drugs and lead in contaminated FMs, whilst providing characteristic and unique fragmentation patterns for each drugs in the FM. The influence of environmental factors such as temperature on the stability of the exogenous substances in FM was investigated by exposing the contaminated FM to different temperatures over 24 h, the findings revealed the drugs' instability at high temperatures and undergo different degrees of degradation whereas lead was more resilient to thermal stress. The detection of triprolidine, metoprolol and sulfamethoxazole from pharmaceutical tablets in FM was successfully achieved by gently touching the tablet powder. The limit of detection (LOD) values of the drugs in the tablet forms were 50, 200 and 750 ng mL−1 for triprolidine, metoprolol and sulfamethoxazole, respectively and their recovery rates were 91.17% for triprolidine, 94.67% for metoprolol and 120.86% for sulfamethoxazole. Finally, to create a genuine casework scenario, contaminated FM was deposited on glass and metal surfaces then subjected to extraction using MCNPs and magnet without compromising the FM impression. Substrate control experiments revealed that the glass surface exhibiting some background signal, however, they did not interfere with the analysis and satisfactory extraction efficiency of endogenous and exogenous components of the FM on both surfaces was obtained using the MCNPs. Overall, this study proves the capability of MCNPs as new SALDI-MS substrate for both extraction and detection of FM components gathering more information pertaining to the donor's lifestyle.

Breast cancer is a global health issue affecting 2.3 million women per year, causing death in over 600,000. Mammography (and biopsy) is the gold standard for screening and diagnosis. Whilst effective, this test exposes individuals to radiation, has limitations to its sensitivity and specificity and may cause moderate to severe discomfort. Some women may also find this test culturally unacceptable. This proof-of-concept study, combining bottom-up proteomics with Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) detection, explores the potential for a non-invasive technique for the early detection of breast cancer from fingertip smears. A cohort of 15 women with either benign breast disease (n = 5), early breast cancer (n = 5) or metastatic breast cancer (n = 5) were recruited from a single UK breast unit. Fingertips smears were taken from each patient and from each of the ten digits, either at the time of diagnosis or, for metastatic patients, during active treatment. A number of statistical analyses and machine learning approaches were investigated and applied to the resulting mass spectral dataset. The highest performing predictive method, a 3-class Multilayer Perceptron (MLP) neural network, yielded an accuracy score of 97.8% when categorising unseen MALDI MS spectra as either the benign, early or metastatic cancer classes. These findings support the need for further research into the use of sweat deposits (in the form of fingertip smears or fingerprints) for non-invasive screening of breast cancer.

The forensic scenario, on which the round robin study was based, simulated a suspected intentional manipulation of a real estate rental agreement consisting of a total of three pages. The aims of this study were to (i) establish the amount and reliability of information extractable from a single type of evidence and to (ii) provide suggestions on the most suitable combination of compatible techniques for a multi-modal imaging approach to forgery detection. To address these aims, seventeen laboratories from sixteen countries were invited to answer the following tasks questions: (i) which printing technique was used? (ii) were the three pages printed with the same printer? (iii) were the three pages made from the same paper? (iv) were the three pages originally stapled? (v) were the headings and signatures written with the same ink? and (vi) were headings and signatures of the same age on all pages? The methods used were classified into the following categories: Optical spectroscopy, including multispectral imaging, smartphone mapping, UV-luminescence and LIBS ; Infrared spectroscopy, including Raman and FTIR (micro-)spectroscopy ; X-ray spectroscopy, including SEM-EDX, PIXE and XPS ; Mass spectrometry, including ICPMS, SIMS, MALDI and LDIMS ; Electrostatic imaging, as well as non- imaging methods, such as non-multimodal visual inspection, (micro-)spectroscopy, physical testing and thin layer chromatography. The performance of the techniques was evaluated as the proportion of discriminated sample pairs to all possible sample pairs. For the undiscriminated sample pairs, a distinction was made between undecidability and false positive claims. It was found that none of the methods used were able to solve all tasks completely and/or correctly and that certain methods were a priori judged unsuitable by the laboratories for some tasks. Correct results were generally achieved for the discrimination of printer toners, whereas incorrect results in the discrimination of inks. For the discrimination of paper, solid state analytical methods proved to be superior to mass spectrometric methods. None of the participating laboratories deemed addressing ink age feasible. It was concluded that correct forensic statements can only be achieved by the complementary application of different methods and that the classical approach of round robin studies to send standardised subsamples to the participants is not feasible for a true multimodal approach if the techniques are not available at one location.

Over the past seven years Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Profiling (MALDI MSP) and Imaging (MALDI MSI) have proven to be feasible tools for the detection of blood and its provenance in stains and fingermarks. However, whilst this capability as a confirmatory test addresses the primary questions at the scene of a violent crime, additional intelligence recoverable from blood can also prove important for investigations. A DNA profile is the most obvious and important example of such intelligence; however, it is not always suitable for identification purposes, depending on quantity, age and environmental conditions. Proteins are much more stable and determining the presence of haemoglobin variants in blood recovered at a crime scene may provide associative and possibly corroborating evidence on the presence of an individual at a particular location. This evidence gains more incriminatory value, the lower the incidence of the variant in a certain geographical area or population and may contribute to narrowing down the pool of suspects. In this study, a MALDI based mass spectrometric method has been developed and tested on six haemoglobin variants for their fast and reliable identification and mapping in blood fingermarks.

Time since deposition is a type of intelligence enabling to address activity level propositions. In relation to a fingermark, this information would provide an additional evidential value by proving/disproving the presence of a suspect at the time of the crime. Whilst fundamentally important, this type of intelligence remains, to date, elusive to the forensic community. This is due to the significant number and interplay of factors that can affect the determination of age. Mass spectrometry (MS) has greatly contributed to the understanding of the changes occurring within the molecular composition of a fingermark over time, especially with regard to lipids. In particular, the hyphenation of chromatography with mass spectrometry has enabled to analyse and monitor over time the complexity of the molecular lipid composition for both volatile and non-volatile species. However, these approaches require extraction of the fingermark molecular components, and as such they are completely destructive. Conversely, chemical imaging methods allow to keep the fingermark intact by enabling in situ analysis. In addition, due to the very nature of mass spectrometry imaging, these methods offer contextual intelligence, that is, time since deposition (potentially) and biometric information for suspect identification; this is particularly desirable when conventional fingermark enhancement methods yield partial, scarce or no ridge detail.

There is evidence that the use of fingerprints for the identification of an individual, either for civil or criminal purposes, has been considered in some form for over 2000 years (Barnes, 2011). The comparison of a mark left by an individual at a crime scene with sets of reference prints taken under controlled conditions is a cornerstone of forensic investigation, first being proposed in 1880 (Faulds, 1880). Following the generation of classification systems for fingermark patterns (Galton, 1892) and filing systems enabling databases to be searched (Henry, 1901), fingerprint comparison and identification has been successfully employed for over 120 years. The fact that fingerprint patterns are ‘unique’ (to the best of scientific knowledge) and persistent throughout life makes them a powerful identification tool. Indeed, the term ‘fingerprint’ is widely used across many other branches of science to describe something that is characteristic and easily distinguished from (e.g.) spectra of other nominally similar substances. In criminal investigations the focus is on locating marks that may have been left by the suspect at the crime scene. These can be of three principal types; a positive mark where material is transferred from the fingertip to the surface, a negative mark where the fingertip removes material (e.g. dust) from the surface, or a ‘plastic’ mark where the fingertip leaves a permanent impression in a soft substance such as putty. The classification of a positive mark can be further divided into a ‘patent’ mark where the material transferred is readily visible to the eye (e.g. mud, ink), or a ‘latent’ mark where the material transferred cannot typically be readily seen by eye and needs further enhancement to be seen. The chemistry of the material transferred from the finger to the surface is important to the visualisation of the contact trace. This is because chemical substances present can be utilised by a range of processes that either convert a latent mark into one that is visible or assist in further enhancing the pre-existing detail in a patent mark. The chemical development of fingermarks was observed as early as the 1860s (Quinche & Margot, 2010) and was already being explored in a more focused way in the 1920s (Mitchell, 1920), with a range of chemical processes targeting different constituents being proposed for use. This review will focus on the material that is ultimately transferred from the fingertip to the surface to form a fingermark. It will consider how this highly complex chemistry can be utilised by a wide range of chemical reagents, and by advanced analytical techniques in combination with imaging capabilities, to reveal the fingermark ridge detail and additional information contained within it.

The reliable identification of blood, as well as the determination of its origin (human or animal) is of great importance in a forensic investigation. Whilst presumptive tests are rapid and deployed in situ, their very nature requires confirmatory tests to be performed remotely. However, only serological tests can determine blood provenance. The present study improves on a previously devised Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS)—proteomics based method for the reliable detection of blood by enabling the determination of blood provenance. The overall protocol was developed to be more specific than presumptive tests and faster/easier than the gold standard liquid chromatography (LC) MS/MS analysis. This is considered a pre-validation study that has investigated stains and fingermarks made in blood, other biofluids and substances that can elicit a false-positive response to colorimetric or presumptive tests, in a blind fashion. Stains and marks were either untreated or enhanced with a range of presumptive tests. Human and animal blood were correctly discriminated from other biofluids and non-biofluid related matrices; animal species determination was also possible within the system investigated. The procedure is compatible with the prior application of presumptive tests. The refined strategy resulting from iterative improvements through a trial and error study of 56 samples was applied to a final set of 13 blind samples. This final study yielded 12/13 correct identifications with the 13th sample being correctly identified as animal blood but with no species attribution. This body of work will contribute towards the validation of MALDI MS based methods and deployment in violent crimes involving bloodshed.

In the last decade, Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) has proven to be a valuable analytical tool in forensic research as it can detect and map molecular information of forensic relevance in trace evidence such as fingermarks and hair. The first published proof of concept demonstrating that it was possible to differentiate males and females from the peptide and protein content of their fingermarks was published in 2012. In that work, MALDI MS was used in Profiling mode (MALDI MSP) to quickly obtain spectral profiles of ungroomed marks. These were submitted to Partial Least Square Discriminant Analysis (PLS-DA) yielding sex discrimination with an accuracy between 67.5% and 74.4%, if harsh classification criteria were applied. Since then, this research has progressed to investigate the opportunity to increase the accuracy of prediction in natural marks (obtained with no preparation of the fingertip prior to deposition) either unenhanced or enhanced prior to matrix application and MALDI analysis. Extensive statistical modelling has been employed to determine the model with the highest sex predictive accuracy. Results show that in natural marks the presence of polymers (as external contaminants) in fingermarks affects the peptide/protein signals to various degrees and, only, by using one type of scoring system, a method has been identified to provide up to 86.1% predictive power in discriminating female from male marks.

In Digital Forensics science, face detection and recognition techniques are nowadays tightly connected. Investigators need accurate results delivered quickly for their official reports, in order to solve a case. Hardware implementation of face detection and recognition systems have shown to be very efficient with high recognition rate and short processing time. FPGAs are used for such implementations, because of the parallelism and the speed they can offer. In this work, we present the overview and the objectives of the MULTI-FORESEE COST project in this area. We also present alternative face detection FPGA implementations, and present face recognition techniques, for hardware integration. Finally, we compare these implementations with respect to the FPGA resources which are currently used. Efficient and flexible solutions are proposed in relation to computing power, performance, and allocated resources.

Latent fingermarks are routinely visualised by subjecting them to one or more CSI/crime lab processes to maximise the recovery of ridge flow and minutiae permitting an identification. In the last decade mass spectrometric imaging (MSI) techniques have been applied to fingermarks to provide information about a suspect and/or on the circumstances of the crime as well as yielding additional images of the ridge pattern. In some cases, these techniques have shown the ability to provide further ridge detail, "filling in the gaps" of the developed mark. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) is presently the most advanced of the so-called ‘surface analysis’ techniques, in terms of compatibility with a number of fingermark enhancement processes and implementation in operational casework. However, for the use of this technique in major crimes to become widespread, compatibility with sequential processing must be demonstrated. This short study has assessed compatibility with a number fingermark processing sequences applied to natural marks on the adhesive side of brown (parcel) and clear tapes. Within the study undertaken, the results confirm the possibility to use MALDI MSI in sequence with multiple processes offering in some instances, complementary ridge detail with respect to that recovered from marks developed by conventional sequence processing.

Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling and Imaging (MALDI MSP and MALDI MSI), in combination with bottom up proteomics, have proven to successfully detect and map blood-derived peptide signatures in blood fingermarks, with high specificity and compatibility with a number of blood enhancement techniques (BET). In the present study, the application of MALDI MSP and MSI to blood marks has been investigated further. In particular, the MALDI based detection and visualisation of blood has been explored in tandem with DNA typing. This investigation has been undertaken in a scenario simulating blood fingermarks on painted walls. In the present study, two sets of marks were analysed with each set comprising of a depletion series of four marks deposited on a surface treated to simulate painted walls: Set I - developed with Ninhydrin (NIN) and Set II- developed with Acid Black-1 (AB-1). For both sets, the application of MALDI MSP was successful in detecting haem and human specific haemoglobin peptide markers. MALDI MSI also provided molecular images by visualising haem on the ridge pattern enhanced by BET. The feasibility of successful and subsequent DNA profiling from the recovered fingermarks was also assessed for marks that had undergone enzymatic in situ digestion and MALDI MSI; it was observed that in 73% of the samples analysed, a DNA profile suitable for comparison was obtained. Based on these results, a possible operational workflow has been proposed incorporating the use of a MALDI MS based approach as a confirmatory test for human blood enabling subsequent DNA typing.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) has been employed to rapidly screen longitudinally sectioned drug user hair samples for cocaine and its metabolites using continuous raster imaging. Optimization of the spatial resolution and raster speed were performed on intact cocaine contaminated hair samples. The optimized settings (100 × 150 μm at 0.24 mm/s) were subsequently used to examine longitudinally sectioned drug user hair samples. The MALDI-MS/MS images showed the distribution of the most abundant cocaine product ion at m/z 182. Using the optimized settings, multiple hair samples obtained from two users were analyzed in approximately 3 h: six times faster than the standard spot-to-spot acquisition method. Quantitation was achieved using longitudinally sectioned control hair samples sprayed with a cocaine dilution series. A multiple reaction monitoring (MRM) experiment was also performed using the ‘dynamic pixel’ imaging method to screen for cocaine and a range of its metabolites, in order to differentiate between contaminated hairs and drug users. Cocaine, benzoylecgonine, and cocaethylene were detectable, in agreement with analyses carried out using the standard LC-MS/MS method. Graphical Abstractᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-017-1766-0) contains supplementary material, which is available to authorized users.

Since the birth of proteomics science in the 1990, the number of applications and of sample preparation methods has grown exponentially, making a huge contribution to the knowledge in life science disciplines. Continuous improvements in the sample treatment strategies unlock and reveal the fine details of disease mechanisms, drug potency, and toxicity as well as enable new disciplines to be investigated such as forensic science. This chapter will cover the most recent developments in sample preparation strategies for tissue proteomics in three areas, namely, cancer, toxicology, and forensics, thus also demonstrating breath of application within the domain of health and well-being, pharmaceuticals, and secure societies. In particular, in the area of cancer (human tumor biomarkers), the most efficient and multi-informative proteomic strategies will be covered in relation to the subsequent application of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid extraction surface analysis (LESA), due to their ability to provide molecular localization of tumor biomarkers albeit with different spatial resolution. With respect to toxicology, methodologies applied in toxicoproteomics will be illustrated with examples from its use in two important areas: the study of drug-induced liver injury (DILI) and studies of effects of chemical and environmental insults on skin, i.e., the effects of irritants, sensitizers, and ionizing radiation. Within this chapter, mainly tissue proteomics sample preparation methods for LC-MS/MS analysis will be discussed as (i) the use of LC-MS/MS is majorly represented in the research efforts of the bioanalytical community in this area and (ii) LC-MS/MS still is the gold standard for quantification studies. Finally, the use of proteomics will also be discussed in forensic science with respect to the information that can be recovered from blood and fingerprint evidence which are commonly encountered at the scene of the crime. The application of proteomic strategies for the analysis of blood and fingerprints is novel and proteomic preparation methods will be reported in relation to the subsequent use of mass spectrometry without any hyphenation. While generally yielding more information, hyphenated methods are often more laborious and time-consuming; since forensic investigations need quick turnaround, without compromising validity of the information, the prospect to develop methods for the application of quick forensic mass spectrometry techniques such as MALDI-MS (in imaging or profiling mode) is of great interest.

The determination of the type of deposition mechanism of blood within fingermarks at the scene of violent crimes is of great importance for the reconstruction of the bloodshed dynamics. However, to date, evaluation still relies on the subjective visual examination of experts. Practitioners encounter three types of scenarios in which blood may be found in fingermarks and they refer to the following three deposition mechanisms: (i) blood marks, originating from a bloodied fingertip; (ii) marks in blood, originating from a clean fingertip contacting a blood contaminated surface; (iii) coincidental deposition mechanisms, originating from a clean fingertip contacting a clean surface, leaving a latent fingermark, and subsequent contamination with blood. The authors hypothesised that, due to differences in distribution of blood in the furrows and on the ridges, the height of blood depositions on the ridges and furrows (and their relative proportions), will differ significantly across the three depositions mechanisms. A second hypothesis was made that the differences would be significant and consistent enough to exploit their measurement as a quantitative and objective way to differentiate the deposition mechanisms. In recent years, infinite focus microscopy (IFM) has been developed, allowing for the computational generation of a 3D image of the topology of a sample via acquisition of images on multiple focal planes. On these bases, it was finally hypothesised that the application of this technique would allow the distinction of deposition mechanisms (i) to (iii). A set of preliminary experiments were designed to test whether IFM was “fit for purpose” and, subsequently, to test if any of the three deposition mechanisms scenarios could be differentiated. Though IFM enabled the analysis of tape lifted samples with some success, for samples produced and analysed directly on the surface of deposition, the results show that the measurements from any scenario will be highly dependent on the original surface of deposition (both in terms of its nature and of the variable exposure to environment); as crime scenes exhibit a wide range of possible relevant surfaces of deposition, the technique showed to not have the desired wide appeal for inclusion into a standardised set of protocols within a routine crime scene workflow.

The sample preparation method reported in this work has permitted for the first time the application of Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling and Imaging (MALDI-MSP and MALDI-MSI) for the detection and mapping of cannabinoids in a single hair sample. MALDI-MSI analysis of hair samples has recently been suggested as an alternative technique to traditional methods of GC-MS and LC-MS due to simpler sample preparation, the ability to detect a narrower time frame of drug use and a reduction in sample amount required. However, despite cannabis being the most commonly used illicit drug worldwide, a MALDI-MS method for the detection and mapping of cannabinoids in a single hair has not been reported. This is probably due to the poor ionization efficiency of the drug and its metabolites and low concentration incorporated into hair. This research showed that the in situ derivatization of cannabinoids through addition of an N-methylpyridium group resulted in improved ionization efficiency, permitting both detection and mapping of Δ9-tetrahydrocannabinol (THC), Cannabinol (CBN), cannabidiol (CBD) and the metabolites 11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH), 11-Hydroxy-delta-9-tetrahydrocannabinol (11-OH-THC) and 11-nor-delta(9)-carboxy-tetrahydrocannabinol glucuronide (THC-COO-glu). Additionally, for the first time an in-source re-arrangement of THC was observed and characterised in this paper thus contributing to new and accurate knowledge in the analysis of this drug by MALDI mass spectrometry.

A bottom up in situ proteomic method has been developed enabling the mapping of multiple blood signatures on the intact ridges of blood fingermarks byMatrix Assisted Laser Desorption Mass Spectrometry Imaging (MALDI-MSI). This method, at a proof of concept stage, builds upon recently published work demonstrating the opportunity to profile and identify multiple blood signatures in bloodstains via a bottom up proteomic approach. The present protocol addresses the limitation of the previously developed profiling method with respect to destructivity;\ud destructivity should be avoided for evidence such as blood fingermarks, where the ridge detail must be preserved in order to provide the associative link between the biometric information and the events of bloodshed. Using a blood mark reference model, trypsin concentration and spraying conditions have been optimised within the technical constraints of the depositor eventually employed; the application of MALDI-MSI and Ion Mobility MS have enabled the detection, confirmation and visualisation of blood signatures directly onto the ridge pattern.\ud These results are to be considered a first insight into a method eventually informing investigations (and judicial debates) of violent crimes in which the reliable and non-destructive detection and mapping of blood in fingermarks is paramount to reconstruct the events of bloodshed.

In the search for better or new methods/techniques to visualise fingermarks or to analyse them exploiting their chemical content, fingermarks inter-variability may hinder the assessment of the method effectiveness. Variability is due to changes in the chemical composition of the fingermarks between different donors and within the same donor, as well as to differential contact time, pressure and angle. When validating a method or comparing it with existing ones, it is not always possible to account for this type of variability. One way to compensate for these issues is to employ, in the early stages of the method development, a device generating reproducible fingermarks. Here the authors present their take on such device, as well as quantitatively describing its performance and benefits against the manual production of marks. Finally a short application is illustrated for the use of this device, at the method developmental stages, in an emerging area of fingerprinting research concerning the retrieval of chemical intelligence from fingermarks.

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence\ud in the forensic investigation of serious offences. Because many of the current enhancement\ud methods used by crime scene investigators are presumptive, the visualisation of blood is not always\ud reliable nor does it bear additional information. In the work presented here, two methods employing a\ud shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols\ud employ both an in solution digestion method and a recently proposed procedure involving immobilization\ud of trypsin on hydrophobin Vmh2 coated MALDI sample plate. The methods are complementary as whilst one yields more identifiable proteins (as biomolecular signatures), the other is extremely rapid (5 minutes).\ud Additionally, data demonstrate the opportunity to discriminate blood provenance even when two different blood sources are present in a mixture. This approach is also suitable for old bloodstains which had been previously chemically enhanced, as experiments conducted on a 9-year-old bloodstain deposited on a ceramic tile demonstrate.

Despite recent improvements to in situ proteolysis strategies, a higher efficiency is still needed to increase both the number of peptides detected and the associated ion intensity, leading to a complete and reliable set of biomarkers for diagnostic or prognostic purposes. In the study presented here, an extract of a systematic study is illustrated investigating a range of surfactants assisting trypsin proteolytic activity. Method development was trialled on fingermarks; this specimen results from a transfer of sweat from an individual’s fingertip to a surface upon contact. As sweat carries a plethora of biomolecules, including peptides and proteins, fingermarks are, potentially, a very valuable specimen for non-invasive prognostic or diagnostic screening. A recent study has demonstrated the opportunity to quickly detect peptides and small proteins in fingermarks using Matrix Assisted Laser Desorption Ionization Mass Spectrometry Profiling (MALDI MSP). However, intact detection bears low sensitivity and does not allow species identification; therefore, a shotgun proteomic approach was employed involving in situ proteolysis. Data demonstrate that in fingermarks, further improvements to the existing method can be achieved using MEGA-8 as surfactant in higher percentages as well as combinations of different detergents. Also, for the first time, Rapigest SF, normally used in solution digestions, has been shown to successfully work also for in situ proteolysis. Graphical Abstract ᅟ Electronic supplementary material The online version of this article (doi:10.1007/s13361-015-1140-z) contains supplementary material, which is available to authorized users.

Latent fingerprints provide a potential route to the secure, high throughput and non-invasive detection of drugs of abuse. In this study we show for the first time that the excreted metabolites of drugs of abuse can be detected in fingerprints using ambient mass spectrometry. Fingerprints and oral fluid were taken from patients attending a drug and alcohol treatment service. Gas chromatography mass spectrometry (GC-MS) was used to test the oral fluid of patients for the presence of cocaine and benzoylecgonine. The corresponding fingerprints were analysed using Desorption Electrospray Ionization (DESI) which operates under ambient conditions and Ion Mobility Tandem Mass Spectrometry Matrix Assisted Laser Desorption Ionization (MALDI-IMS-MS/MS) and Secondary Ion Mass Spectrometry (SIMS). The detection of cocaine, benzoylecgonine (BZE) and methylecgonine (EME) in latent fingerprints using both DESI and MALDI showed good correlation with oral fluid testing. The sensitivity of SIMS was found to be insufficient for this application. These results provide exciting opportunities for the use of fingerprints as a new sampling medium for secure, non-invasive drug detection. The mass spectrometry techniques used here offer a high level of selectivity and consume only a small area of a single fingerprint, allowing repeat and high throughput analyses of a single sample.

Pharmacodynamics and toxicodynamics are the study of the biochemical and physiological effects of therapeutic agents and toxicants and their mechanisms of action. MALDI-MS imaging offers great potential for the study of pharmaco/toxicodynamic responses in tissue owing is its ability to study multiple biomarkers simultaneously in a label-free manner. Here, existing examples of such studies examining anticancer drugs and topically applied treatments are described. Examination of the literature shows that the use of MS imaging in pharmaco/toxicodynamic studies is in fact quite low. The reasons for this are discussed and potential developments in the methodology that might lead to its further use are described.

Since 2009, when Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) was firstly reported for the molecular mapping of latent fingermarks, the range of information and operational capabilities have steadily increased. Pioneering work from our Fingermark Research Group exploited different modalities, including Profiling (MALDI MSP), tandem mass spectrometry (MS/MS) and Ion Mobility MS/MS; a number of methodologies were also developed to conquer a main challenge, namely profiling the suspect and their actions prior to or whilst committing the crime. Suspect profiling here is no longer based on behavioural science but complements this discipline and the investigations by detecting and visualising the molecular make-up of fingermarks onto the identifying ridges. This forensic opportunity provides the link between the biometric information (ridge detail) and the corpus delicti or intelligence on the circumstances of the crime. In 2013, a review was published covering the research work and developments of four years supported by the Home Office, UK, and the local regional Police with some insights (and comparison) into similar research being reported employing other mass spectrometric techniques. The present review is an extensive update on the MALDI MS based methods' achievements, limitations and work in progress in fingermark analysis; it also offers an outlook on further necessary research into this subject. The main highlights are the increased number of possible information retrievable around a suspect and the more extended compatibility of this technology. The latter has allowed MALDI MS based methods to integrate well with current forensic fingerprinting, leading to the investigation of real police casework.

In the study presented here, four examples of crime scene fingermarks analysed by Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI), in collaboration with the local Police Force, are reported. These marks are associated to high profile crimes such as drug dealing, murder and harassment. Following the application of forensic enhancement techniques by the CSI or the crime lab, one of the marks could be directly analysed on the surface of deposition and the others were analysed as primary lifts. In one case, no physical or molecular information was obtained whereas in two cases both ridge detail (albeit not useful for identification purposes) and molecular information could be retrieved. In one case, the intelligence gathered around the suspect's state of mind is, to date the most accomplished demonstration of the benefits and the operational feasibility MALDI MS based methods. These four casework examples are to be considered a first insight into the limitations, benefits and feasibility of MALDI MS based methods in the field; due to the extreme variability in the state of the mark, comprehensive evaluation of these aspects can only be undertaken upon the analysis of a large number of crime scene marks. However, this work does represent a significant advancement, compared to previous published work, as it demonstrates operational feasibility, with different levels of success, despite uncontrollable, unknown and unaccountable environmental and donor variability.

A wide range of fingermark enhancement techniques (FET) is currently employed to visualise latent fingermarks at crime scenes. However, if smudged, partial, distorted or absent in the National Fingerprint Database, crime scene marks may be not useful for identification purposes. In these circumstances, a technology enabling chemical imaging of both endogenous and exogenous species contained within the fingermark could provide additional and associative investigative information, to profile the suspect's activities prior to the crime. Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) has proven to be such a technique, enabling investigative information to be gathered, for example, on what substances the donor has come in contact with and what they have ingested. Nonetheless, to be employed, MALDI MSI has to be validated and its compatibility with FET tested for integration into the standard fingermark examination workflow. For the first time, a direct comparison has been made between the efficiency of a range of FET and MALDI MSI under different conditions. This information will build towards validation of the technology. Also, for the first time, MALDI MSI has been successfully employed as a sequential step following fingermark enhancement using many of the currently employed FET. Additionally, known enhancers have been "re-visited" by combining them with a MALDI matrix, providing both improved fingermark development and chemical species detection via MALDI MSI. The result reported here are good indication in favour of the integration of MALDI MSI into the current fingermark examination workflow for gathering additional investigative information.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has proven to be a powerful analytical tool to investigate problems in several fields of life science. A novel application is in the field of forensics, particularly in the analysis of latent fingermarks. This technology enables images of the fingermark ridge detail and additional intelligence to be simultaneously obtained. Although several methods are available to deposit the MALDI matrix, to make the technology forensically operational, another deposition approach was devised and reported, namely the ‘dry–wet’ method. In the present study, the efficiency of the dry–wet method was evaluated and compared with the conventional spray coat methodology. Results indicate that the dry–wet method is superior for all the donors' typologies in terms of ion signal intensity and clarity of the ridge details. To underpin the reasons of this efficiency, scanning electron microscopy analyses were carried out in parallel to MALDI-MSI experiments using matrices of different particle size. Results have confirmed that the particle size plays an important role in the efficiency of the method as higher quality images and higher intensity spectra are produced as the matrix particle size decreases.

Matrix assisted laser desorption ionisation ion mobility separation mass spectrometry imaging (MALDI-IMS-MSI) has been employed to image the distribution of proteins in ex-vivo human skin. Using a “bottom-up” proteomics approach employing “on-tissue” digestion the distribution of abundant skin proteins; collagen, keratin, decorin and serum albumin could be mapped. Images have been recorded at 150 and 30 μm spatial resolution. Multivariate statistical analysis of the data has been employed to associate specific proteins with layers of the skin. The improved specificity given by the use of ion mobility separation in mass spectrometric imaging has been demonstrated by separation of peptide ions from phospholipids.

Eight years of intensive research have demonstrated that Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Profiling and Imaging (MALDI-MSI and MSP) are powerful tools to gather intelligence around a suspect lifestyle, directly from the identifying ridges of a latent fingermark. In the past three years, many efforts have been invested into translating laboratory methodologies to the field; this was undertaken by devising protocols either for (a) enabling initial fingermark visualisation, such as through the Dry-Wet method, recovery and subsequent MALDI MS based analysis, or for (b) rendering the MS methodologies compatible with the prior application of commonly employed fingermark enhancement techniques (FET). In the present work a major point of interest concerned the sample treatment of FET visualised-lifted fingermarks and the subsequent MS performance of primary tape lifted fingermarks ("primary lifts") versus secondary tape lifted fingermarks (recovery from the surface a second time following the initial primary lift). This was necessary since it may not always be possible to obtain primary lifts of marks visualised at crime scenes for remote MALDI-MSP and MSI. The work illustrated here has provided methodological insights into establishing how to best treat a few types of developed marks in preparation for MALDI-MSI when presented as both secondary and primary lifts; it was demonstrated, as expected, that primary lifts generally yield much higher quality chemical/physical information and are therefore crucial to maximise chances of suspect identification and of retrieval of chemical intelligence. When analysing secondary lifted marks that have been initially developed using aluminium or carbon powders, any of the trialled sample preparation methodologies can be employed except the Dry-Wet method. In the case of TiO2 powder developed marks, the best ridge coverage was achieved by re-enhancing the mark using the initial powder and spray-coating with MALDI matrix. Primary lifts of fingermarks contaminated with an exogenous substance (used as a reference model) yielded the best ridge detail quality whilst for secondary lifts of natural marks pre-enhanced with aluminium powder, significantly greater intensity of the ion image was observed for the sections subjected to either no further enhancement or re-enhancement using aluminium powder.\ud \ud Keywords: Fingermarks; MALDI; Imaging; powders; lifts

Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating disease of the central nervous system (CNS). White matter lesions in MS are surrounded by areas of non-demyelinated normal appearing white matter (NAWM) with complex pathology, including blood brain barrier dysfunction, axonal damage and glial activation. Astrocytes, the most abundant cell type within the CNS, may respond and/or contribute to lesion pathogenesis. We aimed to characterise the transcriptomic profile of astrocytes in NAWM to determine whether specific glial changes exist in the NAWM which contribute to lesion development or prevent disease progression. Astrocytes were isolated from control and NAWM by laser capture microdissection (LCM), using glial fibrillary acidic protein (GFAP) as a marker, and the astrocyte transcriptome determined using microarray analysis. 452 genes were significantly differentially expressed (208 up-regulated and 244 down-regulated, FC ≥ 1.5 and p-value ≤ 0.05). Within the NAWM, astrocytes were associated with significant upregulation of genes involved in the control of iron homeostasis (including metallothionein-1 and -2, ferritin light chain and transferrin), oxidative stress responses, the immune response and neurotrophic support. These findings suggest a neuroprotective role of astrocytes in the NAWM in MS

Matrix-assisted laser desorption/ionization (MALDI), mass spectrometry profiling (MSP), and mass spectrometry imaging (MSI) are continuously pushing the boundaries of many applications in which they can be employed. In the field of forensic science, MALDI MS has proven to be a useful technique but reports were sporadic and only in an academic context until 2009. However, in the past 6 years it has been demonstrated that MALDI MSP and MSI can be practically implemented as an analytical forensic tool in the context of fingermark/fingerprint analysis. Previously always regarded ‘simply’ as physical evidence, fingermarks are an invaluable source of chemical information that can be exploited to provide intelligence on an individual’s lifestyle and activities taking place prior to the crime. A solid proof of principle of the versatility and robustness of MALDI MSP and MSI has gained MALDI a place in the newly launched Fingermark Visualisation Manual edited by the Home Office of the United Kingdom (UK), whose guidelines are followed in the UK and are highly regarded worldwide. Protocols are currently being developed to be used operationally within primarily UK law enforcement. In this chapter, a practical overview is given for informed method development. Protocols that have been successful in answering specific research questions will also be outlined along with the most significant and interesting applications of MALDI MSP and MSI in fingermark/fingerprint analysis.

The first analytical intercomparison of fingerprint residue using equivalent samples of latent fingerprint residue and characterized by a suite of relevant techniques is presented. This work has never been undertaken, presumably due to the perishable nature of fingerprint residue, the lack of fingerprint standards, and the intradonor variability, which impacts sample reproducibility. For the first time, time-of-flight secondary ion mass spectrometry, high-energy secondary ion mass spectrometry, and X-ray photoelectron spectroscopy are used to target endogenous compounds in fingerprints and a method is presented for establishing their relative abundance in fingerprint residue. Comparison of the newer techniques with the more established gas chromatography/mass spectrometry and attenuated total reflection Fourier transform infrared spectroscopic imaging shows good agreement between the methods, with each method detecting repeatable differences between the donors, with the exception of matrix-assisted laser desorption ionization, for which quantitative analysis has not yet been established. We further comment on the sensitivity, selectivity, and practicability of each of the methods for use in future police casework or academic research.

Mass spectrometry imaging (MSI) is a powerful tool for the study of intact tissue sections. The use of matrix-assisted laser desorption/ionisation (MALDI) MSI for the study of the distribution and effect of emollient treatment on sections of reconstructed living skin equivalents during their development and maturation is described. Living skin equivalent (LSE) samples were obtained at 14days development, re-suspended in maintenance medium and incubated for 24h after delivery. The medium was changed, the LSE treated with either Physiogel A.I.® or Oilatum Junior® emollients and then re-incubated and samples taken at 4, 6 and 24h time points. Mass spectra and mass spectral images were recorded from 12μm sections of the LSE taken at each time point for comparison using MALDI mass spectrometry (MS). It was possible to detect ions characteristic of each emollient in the LSE. In addition a number of lipid species previously reported as being significant in the maturation of the LSE were observable. At the 24h time point, the images revealed what appeared to be differences in the organisation of the skin cells observed across the Physiogel A.I.® treatment group tissue sections when directly compared to the untreated tissue group.

Lipidomics is a rapidly expanding area of scientific research and there are a number of analytical techniques that are employed to facilitate investigations. One such technique is matrix-assisted laser desorption ionisation (MALDI) mass spectrometry (MS). Previous MALDI-MS studies involving lipidomic investigation have included the analysis of a number of different ex vivo tissues, most of which were obtained from animal models, with only a few being of human origin. In this study, we describe the use of MALDI-MS, MS/MS and MS imaging methods for analysing lipids within cross-sections of ex vivo human skin. It has been possible to tentatively identify lipid species via accurate mass measurement MALDI-MS and also to confirm the identity of a number of these species via MALDI-MS/MS, in experiments carried out directly on tissue. The main lipid species detected include glycerophospholipids and sphingolipids. MALDI images have been generated at a spatial resolution of 150 and 30 μm, using a MALDI quadrupole time-of-flight Q-Star Pulsar-i TM (Applied Biosystems/MDS Sciex, Concord, ON, Canada) and a MALDI high-definition MS (HDMS) SYNAPT G2-HDMSTM system (Waters, Manchester, UK), respectively. These images show the normal distribution of lipids within human skin, which will provide the basis for assessing alterations in lipid profiles linked to specific skin conditions e.g. sensitisation, in future investigations.

In order to assess the potential of matrix-assisted laser desorption mass spectrometry imaging for the examination of artificial skin models, the absorption of the tricyclic antidepressant imipramine into Straticell-RHE-EPI/001 an artificial model of the human epidermis has been studied. The presence of imipramine could be clearly discerned in treated samples by imaging the distribution of the protonated molecule at m/z 281.18 in samples taken 2 and 8 h after treatment. No clear evidence of biotransformation of imipramine in the artificial epidermal model was detected, although some signals that could potentially be assigned to the desmethyl metabolite were detected. Further work is required in order to investigate the reasons for the apparent low levels of metabolites detected.

An increase in the use of condoms by sexual offenders has been observed. This is likely to be due both to the risk of sexually transmitted diseases and to prevent the transfer of DNA evidence. In this scenario the detection of condom lubricants at a crime scene could aid in proving corpus delicti. Here we show a novel application of Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (MALDI MSI) for mapping the fingermark ridge pattern simultaneously to the detection of the condom lubricant within the fingermark itself. Two condom brands have been investigated to prove the concept. Condoms were handled producing lubricant-contaminated fingermarks. Images of the ridge pattern were obtained simultaneously to the detection of two lubricants, even several weeks after the fingermark deposition. The results therefore show the potential of MALDI MSI to link the suspect (identification through fingermark ridge pattern) to the crime (detection of condom lubricant) in one analysis. This would enable forensic scientists to provide evidence with stronger support in alleged cases of sexual assault.

The aim of our pilot study was to investigate, by a proteomic approach, the expressed differences in cerebrospinal fluid (CSF) protein patterns in order to aid in the diagnosis and treatment of normal pressure hydrocephalus (NPH). Seventeen patients with NPH, selected by Intracranial-Pressure monitoring (ICPmo), underwent implantation of a shunt and after 6 months were clinically re-evaluated. Thirteen patients improved, whereas four did not. During ICPmo CSF was collected and its proteoma was analyzed by 2D gel electrophoresis and mass spectrometry. The over-expression of alpha2HS glycoprotein, alpha1 antichimotrypsin and alpha1beta glycoprotein and the under-expression of glial fibrillary acidic protein, apolipoproteins (AIV, J and E), complement C3c, anti-thrombin, alpha2 antiplasmin and albumin seem to be associated with a positive response to surgery. Most of these proteins have been reported to be altered in Alzheimer disease, supporting the hypothesis of a possible link between these two nosological entities.

Identification of suspects via fingermark analysis is one of the mainstays of forensic science. The success in matching fingermarks, using conventional fingermark scanning and database searching, strongly relies on the enhancement method adopted for fingermark recovery; this in turn depends on the components present in the fingermarks, which will change over time. This work aims to develop a robust methodology for improved analytical detection of the fingermark components. For the first time, matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been used to image endogenous lipids from fresh and aged, groomed and ungroomed fingermarks. The methodology was initially developed using oleic acid which was detected along with its degradation products over a 7-day period, at three different temperatures in a time-course experiment. The optimised methodology was then transferred to the imaging analysis of real fingermark samples. Fingermark patterns were reconstructed by retrieving the m/z values of oleic acid and its degradation products. This allowed the three aged fingermarks to be distinguished. In order to prove that MALDI-MSI can be used in a non-destructive way, a simple washing protocol was adopted which returned a fingermark that could be further investigated with classical forensic approaches. The work reported here proves the potential and the feasibility of MALDI-MSI for the forensic analysis of fingermarks, thus making it competitive with other MSI techniques such as desorption electrospray ionisation (DESI)-MS. The feasibility of using MALDI-MSI in fingermark ageing studies is also demonstrated along with the potential to be integrated into routine fingermark forensic analysis.