Vikki A. Carolan

Novel molecular tumour classification using MALDI–mass spectrometry imaging of tissue micro-array

The development of tissue micro-array (TMA) technologies provides insights into high-throughput analysis of proteomics patterns from a large number of archived tumour samples. In the work reported here, matrix-assisted laser desorption/ionisation–ion mobility separation–mass spectrometry (MALDI–IMS–MS) profiling and imaging methodology has been used to visualise the distribution of several peptides and identify them directly from TMA sections after on-tissue tryptic digestion. A novel approach that combines MALDI–IMS–MSI and principal component analysis–discriminant analysis (PCA–DA) is described, which has the aim of generating tumour classification models based on protein profile patterns. The molecular classification models obtained by PCA–DA have been validated by applying the same statistical analysis to other tissue cores and patient samples. The ability to correlate proteomic information obtained from samples with known and/or unknown clinical outcome by statistical analysis is of great importance, since it may lead to a better understanding of tumour progression and aggressiveness and hence improve diagnosis, prognosis as well as therapeutic treatments. The selectivity, robustness and current limitations of the methodology are discussed.

MALDI-Ion Mobility Separation-Mass Spectrometry Imaging of Glucose-Regulated Protein 78 kDa (Grp78) in Human Formalin-Fixed, Paraffin-Embedded Pancreatic Adenocarcinoma Tissue Sections

MALDI-mass spectrometry imaging (MALDI-MSI) is a technique that allows proteomic information, that is, the spatial distribution and identification of proteins, to be obtained directly from tissue sections. The use of in situ enzymatic digestion as a sample pretreatment prior to MALDI-MSI analysis has been found to be useful for retrieving protein identification directly from formalin-fixed, paraffin-embedded (ffpe) tissue sections. Here, an improved method for the study of the distribution and the identification of peptides obtained after in situ digestion of fppe pancreatic tumor tissue sections by using MALDI-mass spectrometry imaging coupled with ion mobility separation (IMS) is described. MALDI-IMS-MS images of peptide obtained from pancreatic tumor tissue sections allowed the localization of tumor regions within the tissue section, while minimizing the peak interferences which were observed with conventional MALDI-TOF MSI. The use of ion mobility separation coupled with MALDI-MSI improved the selectivity and specificity of the method and, hence, enabled both the localization and in situ identification of glucose regulated protein 78 kDa (Grp78), a tumor biomarker, within pancreatic tumor tissue sections. These findings were validated using immunohistochemical staining.

Direct detection of peptides and small proteins in fingermarks and determination of sex by MALDI mass spectrometry profiling

Matrix Assisted Laser Desorption Ionisation Mass Spectrometry (MALDI MS) can detect and image a variety of endogenous and exogenous compounds from latent fingermarks. This opportunity potentially provides investigators with both an image for suspect identification and chemical information to be used as additional intelligence. The latter becomes particularly important when the fingermark is distorted or smudged or when the suspect is not a previously convicted offender and therefore their fingerprints are not present in the National Fingerprint Database. One of the desirable pieces of intelligence would be the sex of the suspect from the chemical composition of a fingermark. In this study we show that the direct detection of peptides and proteins from fingermarks by MALDI MS Profiling (MALDI MSP), along with the multivariate modeling of the spectra, enables the determination of sex with 85% accuracy. The chemical analysis of the fingermark composition is expected to additionally provide information on traits such as nutritional habits, drug use or hormonal status.

Examination of the distribution of nicosulfuron in sunflower plants by matrix-assisted laser desorption/ionisation mass spectrometry imaging

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been used to image the distribution of the pesticide nicosulfuron (2-[[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]aminosulfonyl]-N,N-dimethyl-3-pyridinecarboxamide) in plant tissue using direct tissue imaging following root and foliar uptake. Sunflower plants inoculated with nicosulfuron were horizontally sectioned at varying distances along the stem in order to asses the extent of translocation; uptake via the leaves following foliar application to the leaves and uptake via the roots from a hydroponics system were compared. An improved sample preparation methodology, encasing samples in ice, allowed sections from along the whole of the plant stem from the root bundle to the growing tip to be taken. Images of fragment ions and alkali metal adducts have been generated that show the distribution of the parent compound and a phase 1 metabolite in the plant. Positive and negative controls have been included in the images to confirm ion origin and prevent false-positive results which could originate from endogenous compounds present within the plant tissue.

The speciation of inorganic and methylmercury in human hair by high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry

Inorganic and methylmercury content in human hair has previously been determined using different analytical techniques for each species. In this study a single method, allowing the separation and determination of mercury species in human hair, is developed. Using a HPLC-ICP-MS system it is possible to separate inorganic and methylmercury in hair, without any modifications to the existing instrumentation. The results showed that in order to determine methylmercury as well as inorganic mercury the hair sample must be cold digested with 2 ml of concentrated nitric acid plus 1 ml of hydrogen peroxide and that a minimum of 0.1 g hair is required. Hair washing procedures are also investigated to remove exogenously bound species. It is seen that when hair is soaked with simulated sweat solution containing both inorganic and methylmercury and then washed with 0.1 M HCl all the methylmercury, both existing and spiked, can be removed from the hair sample. However, only 65% of the spiked inorganic mercury can be removed by washing with 0.1 M HCl, the rest being irreversibly bound to the hair.

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.

Direct analysis of pharmaceutical tablet formulations using matrix‐assisted laser desorption/ionisation mass spectrometry imaging

Matrix-Assisted Laser Desorption/Ionisation Mass Spectrometry Imaging (MALDI MSI) has been used to directly analyse a range of tablets in order to assess the homogeneity of the active drug compound throughout the excipients contained within the tablets studied. The information gained from the imaging experiments can be used to improve and gain a greater understanding of the manufacturing process; such knowledge will enable improvements in finished product quality to make safer and more efficacious tablet formulations. Commercially available and prescription tablet formulations have been analysed, including aspirin, paracetamol, sildenafil citrate (Viagra(R)) and a batch of tablets in development (tablet X: placebo; 1 mg; 3 mg and 6 mg). MALDI MSI provides semi-quantitative information that is related to ion abundance, therefore Principal Component Analysis (PCA), a multivariate analysis technique, has been used to differentiate between tablets containing different amounts of active drug ingredient. Aspects of sample preparation have also been investigated with regard to tablet shape and texture. The results obtained indicate that MALDI MSI can be used effectively to analyse the spatial distribution of the active pharmaceutical component (API) in pharmaceutical tablet formulations.

Characterization of the calcification of cardiac valve bioprostheses by environmental scanning electron microscopy and vibrational spectroscopy

Bioprosthetic heart valve tissue and associated calcification were studied in their natural state, using environmental scanning electron microscopy (ESEM). Energy dispersive X‐ray micro‐analysis, X‐ray diffraction, Fourier‐transform infrared and Raman spectroscopy were used to characterize the various calcific deposits observed with ESEM. The major elements present in calcified valves were also analyzed by inductively coupled plasma–optical emission spectroscopy. To better understand the precursor formation of the calcific deposits, results from the elemental analyses were statistically correlated. ESEM revealed the presence of four broad types of calcium phosphate crystal morphology. In addition, two main patterns of organization of calcific deposits were observed associated with the collagen fibres. Energy dispersive X‐ray micro‐analysis identified the crystals observed by ESEM as salts containing mainly calcium and phosphate with ratios from 1.340 (possibly octacalcium phosphate, which has a Ca/P ratio of 1.336) to 2.045 (possibly hydroxyapatite with incorporation of carbonate and metal ion contaminants, such as silicon and magnesium, in the crystal lattice). Raman and fourier‐transform infrared spectroscopy also identified the presence of carbonate and the analyses showed spectral features very similar to a crystalline hydroxyapatite spectrum, also refuting the presence of precursor phases such as β‐tricalcium phosphate, octacalcium phosphate and dicalcium phosphate dihydrate. The results of this study raised the possibility of the presence of precursor phases associated with the early stages of calcification.

Instrumentation and software for mass spectrometry imaging—Making the most of what you've got

Whilst it might be desirable to be able to purchase an up to date mass spectrometry platform and dedicate it to mass spectrometry imaging, this is not the situation initially for many laboratories. There are a variety of methods by which existing mass spectrometers can be upgraded/adapted to perform mass spectrometry imaging using MALDI, DESI or LAESI as the means of generating ions. The focus of this article is on relatively low cost adaptations of existing instrumentation with suggestions made for performance enhancements where appropriate. A brief description of attempts to perform SIMS imaging on quadrupole time of flight mass spectrometers is also given. The required software is described with particular emphasis on freeware packages which can be used to display/enhance data. Requirements for data pre-processing prior or statistical analysis are discussed along with the use of MATLAB® for the analysis itself.

Examination of the translocation of sulfonylurea herbicides in sunflower plants by matrix-assisted laser desorption/ionisation mass spectrometry imaging.

Pesticides are widely used in agriculture to control weeds, pests and diseases. Successful control is dependent on the compound reaching the target site within the organism after spray or soil application. Conventional methods for determining uptake and movement of herbicides and pesticides include autoradiography, liquid scintillation and chromatographic techniques such as high-performance liquid chromatography (HPLC). Autoradiography using radiolabelled compounds provides the best indication of a compounds movement within the plant system. Autoradiography is an established technique but it relies on the synthesis of radiolabelled compounds. The distribution of four sulfonylurea herbicides in sunflower plants has been studied 24  h after foliar application. The use of matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) images of protonated molecules and fragment ions (resulting from fragmentation at the urea bond within the sulfonylurea herbicides) has provided evidence for translocation above and below the application point. The translocation of nicosulfuron and azoxystrobin within the same plant system has also been demonstrated following their application to the plant stem. This study provides evidence that MALDI-MSI has great potential as an analytical technique to detect and assess the foliar, root and stem uptake of agrochemicals, and to reveal their distribution through the plant once absorbed and translocated.