Sample Treatment for Tissue Proteomics in Cancer, Toxicology, and Forensics.

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.

Investigation of infinite focus microscopy for the determination of the association of blood with fingermarks.

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.

Examination of the skin barrier repair/wound healing process using a living skin equivalent model and matrix-assisted laser desorption-ionization-mass spectrometry imaging.

OBJECTIVE: Examination of the skin barrier repair/wound healing process using a living skin equivalent (LSE) model and matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) to identify lipids directly involved as potential biomarkers. These biomarkers may be used to determine whether an in vivo wound is going to heal for example if infected. METHODS: An in vitro LSE model was wounded with a scalpel blade and assessed at day 4 post-wounding by histology and MALDI-MSI. Samples were sectioned at wound site and were either formalin-fixed paraffin-embedded (FFPE) for histology or snapped frozen (FF) for MSI analysis. RESULTS: The combination of using an in vitro wounded skin model with MSI allowed the identification of lipids involved in the skin barrier repair/wound healing process. The technique was able to highlight lipids directly in the wound site and distinguish differences in lipid distribution between the epidermis and wound site. CONCLUSION: This novel method of coupling an in vitro LSE with MSI allowed in-depth molecular analysis of the skin barrier repair/wound healing process. The technique allowed the identification of lipids directly involved in the skin barrier repair/wound healing process, indicating these biomarkers may be potentially be used within the clinic. These biomarkers will help to determine, which stage of the skin barrier repair/wound healing process the wound is in to provide the best treatment.

Metabolomic analysis of white and yellow seminal plasma in turkeys (Meleagris gallopavo).

Numerous studies have indicated that yellow semen syndrome (YSS) of turkey is associated with the production of low semen quality, resulting in reduced fertility and hatchability. It is unknown at present if the etiology of YSS also could be linked to low-molecular weight metabolites. The aim of this study was to examine the metabolome of white and yellow seminal plasma of turkeys. Two different metabolomics approaches, shotgun (direct infusion) and liquid chromatography-mass spectrometry (LC-MS), were employed to identify metabolites differentially abundant in yellow seminal plasma. Significant changes in the levels of 1549 and 2093 metabolites were detected in yellow vs. white seminal plasma using shotgun and LC-MS, respectively. Of these, 354 metabolites (189 increased and 165 decreased) after shotgun and 936 metabolites (363 increased and 573 decreased) after LC-MS were putatively identified using the Human Metabolome Database. Significantly differentiated metabolites were subjected to Ingenuity Pathway Analysis. Lipid metabolism, molecular transport, and nucleic acid metabolism were the top pathways that differentiated white and yellow seminal plasma. These data strongly suggest that disturbance of carbohydrate and lipid metabolism is characteristic for YSS. The abnormal metabolism of lipids may contribute to the numerous lipid vacuoles previously observed in the reproductive tracts of YSS males. An increased level of riboflavin in YSS may be responsible for yellow turkey semen pigmentation. A disturbance in thyroid hormone metabolism visible at protein and metabolic levels may be involved in YSS in turkey. The low quality of YSS may be linked with the presence of drug residues in the reproductive tract.

A proteomic approach for the rapid, multi-informative and reliable identification of blood.

Blood evidence is frequently encountered at the scene of violent crimes and can provide valuable intelligence in the forensic investigation of serious offences. Because many of the current enhancement methods used by crime scene investigators are presumptive, the visualisation of blood is not always reliable nor does it bear additional information. In the work presented here, two methods employing a shotgun bottom up proteomic approach for the detection of blood are reported; the developed protocols employ both an in solution digestion method and a recently proposed procedure involving immobilization 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). 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.

Direct detection of blood in fingermarks by MALDI MS profiling and imaging.

The determination of the presence of blood in fingermarks constitutes important intelligence in a criminal investigation as it helps to reconstruct the events that have taken place at a scene of crime. Various methodologies have been reported and are currently employed for the detection of the presence of blood including optical, spectroscopic and chemical development approaches. However, most methods only give an indication that blood may be present and, therefore, these methods are described as presumptive tests. Here we show the use of Matrix-Assisted Laser Desorption Ionisation Mass Spectrometry Profiling and Imaging (MALDI MSP and MALDI MSI) for the determination of the presence of blood in fingermarks by specifically detecting the molecules of haem and haemoglobin through their mass-to-charge ratios. Furthermore, preliminary experiments are shown which demonstrate that this technology is compatible with other methods currently employed for enhancing fingermarks in blood (or contaminated by blood). The application of the developed protocols to a crime scene blood trace, demonstrates the feasibility of using this technology in routine casework. These findings open up a new line of research for the development of robust MALDI MSP and MALDI MSI protocols for the detection and chemical imaging of bloodied marks.

Beyond the ridge pattern: multi-informative analysis of latent fingermarks by MALDI mass spectrometry.

After over a century, fingerprints are still one of the most powerful means of biometric identification. The conventional forensic workflow for suspect identification consists of (i) recovering latent marks from crime scenes using the appropriate enhancement technique and (ii) obtaining an image of the mark to compare either against known suspect prints and/or to search in a Fingerprint Database. The suspect is identified through matching the ridge pattern and local characteristics of the ridge pattern (minutiae). However successful, there are a number of scenarios in which this process may fail; they include the recovery of partial, distorted or smudged marks, poor quality of the image resulting from inadequacy of the enhancement technique applied, extensive scarring/abrasion of the fingertips or absence of suspect's fingerprint records in the database. In all of these instances it would be very desirable to have a technology able to provide additional information from a fingermark exploiting its endogenous and exogenous chemical content. This opportunity could potentially provide new investigative leads, especially when the fingermark comparison and match process fails. We have demonstrated that Matrix Assisted Laser Desorption Ionisation Mass Spectrometry and Mass Spectrometry Imaging (MALDI MSI) can provide multiple images of the same fingermark in one analysis simultaneous with additional intelligence. Here, a review on the pioneering use and development of MALDI MSI for the analysis of latent fingermarks is presented along with the latest achievements on the forensic intelligence retrievable.

Curcumin: a multipurpose matrix for MALDI mass spectrometry imaging applications.

Curcumin, 1,7-bis-(4-hydroxy-3-methoxy-phenyl)-hepta-1,6-diene-3,5-dione, is a polyphenolic compound naturally present in the Curcuma longa plant, also known as tumeric. Used primarily as a coloring agent and additive in food, curcumin has also long been used for its therapeutic properties in a number of medical scenarios. Here, we report on an entirely novel use of curcumin; its extended structure of conjugated double bonds suggested the potential of this compound to be a good matrix assisted laser desorption ionization mass spectrometry (MALDI MS) matrix candidate. In the quest for novel and more efficient MALDI MS matrices, curcumin is revealed to be a versatile and multipurpose matrix. It has been applied successfully for the analysis of pharmaceuticals and drugs, for imaging lipids in skin and lung tissues, and for the analysis of a number of compound classes in fingermarks. In each case, the use of curcumin is shown to promote analyte ionization very efficiently as well as provide excellent mass spectral image quality.

Separation of overlapping fingermarks by matrix assisted laser desorption ionisation mass spectrometry imaging.

Latent fingermarks are impressions of the skin ridge pattern that are transferred by the accidental contact of fingertips with a deposition surface. The ability to enhance, lift and produce an image of a latent fingermark, for comparison and suspect match against a central fingerprint database, provides forensic investigators with what is still considered one of the most powerful means of biometric identification to date. Identification relies on the recovery, visualisation, extraction and comparison of local characteristics of the ridge pattern (minutiae) that are unique to individuals. Therefore, both for manual inspection of the minutiae and using automated ridge extraction algorithms, the clearer the ridge details, the more reliable and successful the match. Overlapping fingermarks pose a remarkable challenge in this context and are often encountered when developing marks from crime scenes. Here we propose the use of Matrix Assisted Laser Desorption Ionisation Mass Spectrometry Imaging (MALDI MSI) to separate overlapping fingermarks using ion signals that are characteristic of each fingermark and that may be endogenous or exogenous in nature. In this work we show that the methodology works in a number of different scenarios both using manual inspection of the spectrum profile or a much quicker multivariate statistical analysis.

Investigation of protein induction in tumour vascular targeted strategies by MALDI MSI.

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.

Variations in the estimation of the contribution of environmental tobacco smoke (ETS) to respirable (< or = 5 microns) indoor air particulates obtained by the use of different analytical methods.

Several methods are in use for the identification of the contribution of particulate associated environmental tobacco smoke (ETS) and sidestream smoke to an atmosphere. These include the measurement of respirable suspended particulates (RSP), measurements of the total UV absorption and total fluorescence emission of a methanol extract of collected particulates and the use of specific marker compounds such as solanesol and scopoletin. Use of these methods gave values for the contribution of particulate ETS to total respirable (< or = 5 microns) particulates in the ranges 8.3-124.7% for smokers' houses and 9.6-121.2% for smokers' offices, respectively. However, using what we consider to be the most reliable methodology, based on the measurement of solanesol, the average contribution of particulate ETS to total respirable (< or = 5 microns) particulates for smokers' houses was 21.7% and for smokers' offices was 23.3%.

Glutaraldehyde-induced cross-links: a study of model compounds and commercial bioprosthetic valves.

BACKGROUND AND AIM OF THE STUDY: The treatment of bioprosthetic tissue routinely involves the use of glutaraldehyde, although the specific chemistry of glutaraldehyde fixation is not fully understood. Descriptions of definitive work on this reaction using model compounds are limited. The aim of the present study was to increase our understanding of the chemistry involved in the treatment of collagen-rich tissue with glutaraldehyde. Initially, 6-aminohexanoic acid (6-AHA) was used to model the lysine/hydroxylysine molecules in collagen before studying the more complex chemistry of the tissue. METHODS: The reaction between 0.6% glutaraldehyde and 6-AHA was studied by positive ion electrospray-mass spectroscopy. Untreated, locally treated and commercially produced explanted and non-implanted tissue were hydrolyzed under various conditions and analyzed both directly and after derivatization with 4-chlorophenylhydrazine, 4-bromophenacyl bromide and dansyl chloride by reverse-phase-high performance liquid chromatography-mass spectrometry. RESULTS: The mass spectral data obtained from the reaction of glutaraldehyde with 6-AHA showed the presence of alpha,beta unsaturated aldehydes and their further condensation products involving Michael reactions of glutaraldehyde, Schiff base cross-links and various cyclization products incorporating pyridinium and dihydropyridine ring structures. The only stable cross-link detected was an 'anabilysine'-like compound. Similar structures were present in the tissue, and anabilysine was identified by tandem mass spectrometry. CONCLUSION: The results from the reaction of glutaraldehyde with 6-AHA agree with those published previously. The only detectable stable cross-link definitively identified in treated bioprosthetic tissue was anabilysine. No long-chain polymers of glutaraldehyde were detected.

Influence of surface carbon coverage of C1 (TMS) stationary phases on the separation of nonylphenol ethoxylate ethoxymers.

The determination of surfactants in surface waters is required owing to their toxicity to aquatic micro-organisms and potential as endocrine disrupters. We have previously reported a method for the simultaneous separation of linear alkyl benzene sulfonates (LAS) and nonylphenol ethoxylates (NPEO) by high-performance liquid chromatography using a C1 (TMS) column. In this earlier work we discussed some problems with the resolution of individual ethoxymers from NPEO using C1 columns from different manufacturers. Here, we postulate that this phenomenon may be linked to carbon coverage of the C1 (TMS) stationary phases and study this utilising both elemental (bulk) analyses and surface specific analyses by X-ray photoelectron spectroscopy. Data obtained indicate that for the simultaneous separation of the LAS homologues and ethoxymers of NPEO, the stationary phase must have some trimethylsilyl groups bound to the surface of the silica in order to achieve separation of the LAS homologues, however the degree of surface coverage must not be greater than ca. 0.5 micromol/m2 in order to achieve adequate resolution of the NPEO ethoxymers. These data support earlier evidence for a "pseudo" reversed-phase mechanism for this separation.

Analysis of urinary organic acids by Plasmaspray liquid chromatography/mass spectrometry.

High-performance liquid chromatography is being used increasingly as a screening method to detect organic acid-urias, an important group of inherited metabolic disorders. Analysis is hampered by lack of a suitable specific detection system. We have carried out preliminary investigations to assess the potential value of liquid chromatography/mass spectrometry with a plasmaspray interface. Spectra of standard acids yielded intense [M-H]- ions with little fragmentation. Organic acids could not be identified in urine samples from healthy neonates because of poor sensitivity. However, urine from a baby with the inherited disorder methylmalonic aciduria showed a distinct peak of methylmalonic acid, easily identified owing to its high sample concentration.