Detection and imaging of pharmaceutical compounds in skin by MALDI-MS.

Mass spectrometric techniques have been developed recently for the examination of biological tissue samples. Tandem mass spectrometry has been employed for the detection of pharmaceutical compounds and also mass spectrometric 'images' have been produced which show the spatial distribution of peptides, proteins and drugs in tissue.In this thesis, a programme of method development for the detection and imaging of topically applied pharmaceutical compounds in porcine epidermal tissue by MALDI-TOF-MS is presented. Direct analysis of fresh tissue sections was compared with the analysis of tissue imprints formed by blotting onto a variety of substrates. The samples were coated with matrix material by a prototype electrospray deposition device. Analyses were performed on a linear time-of-flight (LaserTOF 1500, SAI) mass spectrometer. Direct analysis of tissue and analysis of the C18 blots gave irreproducible data. Problems with matrix layer in-homogeneity were experienced with nitrocellulose and polyvinyl difluoride (PVDF) membranes. Reproducible data were obtained by analysis of tissue imprints created on carbon and cellulose membranes. All subsequent work was conducted using an Applied Biosystem Qstar pulsar i hybrid quadrupole time-of-flight mass spectrometer fitted with an orthogonal MALDI ion source and ion imaging software. The advantages of superior mass accuracy and resolution with such an instrument configuration were investigated.Electrospray and airspray methods were compared for analysis of tissue imprinted carbon and cellulose membranes. A novel method of pre-coating cellulose membranes in matrix by airspray prior to the blotting procedure was developed. The method was found to retain the expected distribution of the analyte.Ion images demonstrating the permeation of the applied compound into the skin were achieved by imaging a cross sectional imprint of treated tissue on a cellulose membrane precoated in matrix material. A calibration graph for the determination of ketoconazole was prepared using the sodium adduct of the matrix ion as an internal standard. This enabled construction of a quantitative profile of drug in skin. Conventional haematoxylin and eosin staining and microscopy methods were employed to obtain a histological image of the porcine epidermal tissue. Super imposing the mass spectrometric and histological images revealed drug permeation into the dermal tissue layer. A quantitative corneum tape stripping/HPLC method was developed for comparison. Useful data was acquired and further work suggested to facilitate a full validation of the methods presented in this thesis

The use of random projections for the analysis of mass spectrometry imaging data

The ‘curse of dimensionality’ imposes fundamental limits on the analysis of the large, information rich datasets that are produced by mass spectrometry imaging. Additionally, such datasets are often too large to be analyzed as a whole and so dimensionality reduction is required before further analysis can be performed. We investigate the use of simple random projections for the dimensionality reduction of mass spectrometry imaging data and examine how they enable efficient and fast segmentation using k-means clustering. The method is computationally efficient and can be implemented such that only one spectrum is needed in memory at any time. We use this technique to reveal histologically significant regions within MALDI images of diseased human liver. Segmentation results achieved following a reduction in the dimensionality of the data by more than 99% (without peak picking) showed that histologic changes due to disease can be automatically visualized from molecular images. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-014-1024-7) contains supplementary material, which is available to authorized users

Dried blood spot proteomics : surface extraction of endogenous proteins coupled with automated sample preparation and mass spectrometry analysis

Dried blood spots offer many advantages as a sample format including ease and safety of transport and handling. To date, the majority of mass spectrometry analyses of dried blood spots have focused on small molecules or hemoglobin. However, dried blood spots are a potentially rich source of protein biomarkers, an area that has been overlooked. To address this issue, we have applied an untargeted bottom-up proteomics approach to the analysis of dried blood spots. We present an automated and integrated method for extraction of endogenous proteins from the surface of dried blood spots and sample preparation via trypsin digestion by use of the Advion Biosciences Triversa Nanomate robotic platform. Liquid chromatography tandem mass spectrometry of the resulting digests enabled identification of 120 proteins from a single dried blood spot. The proteins identified cross a concentration range of four orders of magnitude. The method is evaluated and the results discussed in terms of the proteins identified and their potential use as biomarkers in screening programs. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-013-0658-1) contains supplementary material, which is available to authorized users

Comparison of Oral, Intranasal and Aerosol Administration of Amiodarone in Rats as a Model of Pulmonary Phospholipidosis.

‘Foamy’ alveolar macrophages (FAM) observed in nonclinical toxicology studies during inhaled drug development may indicate drug-induced phospholipidosis, but can also derive from adaptive non-adverse mechanisms. Orally administered amiodarone is currently used as a model of pulmonary phospholipidosis and it was hypothesized that aerosol administration would produce phospholipidosis-induced FAM that could be characterized and used in comparative inhalation toxicology. Han-Wistar rats were given amiodarone via (1) intranasal administration (6.25 mg/kg) on two days, (2) aerosol administration (3 mg/kg) on two days, (3) aerosol administration (10 mg/kg) followed by three days of 30 mg/kg or (4) oral administration (100 mg/kg) for 7 days. Alveolar macrophages in bronchoalveolar lavage were evaluated by di_erential cell counting and high content fluorescence imaging. Histopathology and mass-spectrometry imaging (MSI) were performed on lung slices. The higher dose aerosolised amiodarone caused transient pulmonary inflammation (p < 0.05), but only oral amiodarone resulted in FAM (p < 0.001). MSI of the lungs of orally treated rats revealed a homogenous distribution of amiodarone and a putative phospholipidosis marker, di-22:6 bis-monoacylglycerol, throughout lung tissue whereas aerosol administration resulted in localization of both compounds around the airway lumen. Thus, unlike oral administration, aerosolised amiodarone failed to produce the expected FAM responses.Peer reviewedFinal Published versio

Top-down and bottom-up identification of proteins by liquid extraction surface analysis mass spectrometry of healthy and diseased human liver tissue

Liquid extraction surface analysis mass spectrometry (LESA MS) has the potential to become a useful tool in the spatially-resolved profiling of proteins in substrates. Here, the approach has been applied to the analysis of thin tissue sections from human liver. The aim was to determine whether LESA MS was a suitable approach for the detection of protein biomarkers of nonalcoholic liver disease (nonalcoholic steatohepatitis, NASH), with a view to the eventual development of LESA MS for imaging NASH pathology. Two approaches were considered. In the first, endogenous proteins were extracted from liver tissue sections by LESA, subjected to automated trypsin digestion, and the resulting peptide mixture was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) (bottom-up approach). In the second (top-down approach), endogenous proteins were extracted by LESA, and analyzed intact. Selected protein ions were subjected to collision-induced dissociation (CID) and/or electron transfer dissociation (ETD) mass spectrometry. The bottom-up approach resulted in the identification of over 500 proteins; however identification of key protein biomarkers, liver fatty acid binding protein (FABP1), and its variant (Thr→Ala, position 94), was unreliable and irreproducible. Top-down LESA MS analysis of healthy and diseased liver tissue revealed peaks corresponding to multiple (~15–25) proteins. MS/MS of four of these proteins identified them as FABP1, its variant, α-hemoglobin, and 10 kDa heat shock protein. The reliable identification of FABP1 and its variant by top-down LESA MS suggests that the approach may be suitable for imaging NASH pathology in sections from liver biopsies. [Figure: see text] ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s13361-014-0967-z) contains supplementary material, which is available to authorized users