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TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS ©2017 Waters Corporation | LL-pdf ADVANCES IN MULTI-MODAL MASS SPECTROMETRY IMAGING FOR BIOMEDICAL APPLICATIONS 1 Roy Martin, Emrys Jones 2 , Philippa Hart 2 , Mark Towers 2 , James Langridge 2 , Emmanuelle Claude 2 1 Waters Corporation, Beverly, MA USA, 2 Waters Corporation, Wilmslow, United Kingdom INTRODUCTION Mass spectrometry imaging (MSI) is now increasingly used for clinical research applications due to significant technological improvements. Matrix-assisted laser desorption/ionization (MALDI), initially introduced by Caprioli et al 1 , is the dominant MSI technique used today, due to the ability to analyse intact proteins directly from tissue and has been commercially developed by a number of vendors. In the last few years, desorption electrospray ionization (DESI) has been developed and can ionize clinically important molecules such as lipids directly from tissue. DESI, a surface analysis technique incorporating an electrospray probe, can be utilized as a spatially resolved imaging technique by rastering a surface under the spray using a high precision XY stage. As the electrospray droplets impact upon a surface, chemical constituents are desorbed and transferred into the atmospheric inlet of the mass spectrometer source. Ionization occurs due to the charge imparted onto the droplets. No modification to the sample such as matrix addition is necessary and therefore minimum sample preparation is required to run a DESI imaging experiment, making this technique more compatible within a clinical research environment. Here we compare and contrast MALDI and DESI imaging techniques which are operated on an oA-QTOF mass spectrometer. METHODS Sample preparation The tissue sections analyzed were frozen rodent brain and clinical human tissue sections. MALDI experiments were carried conducted by spraying a matrix solution was using a commercial SunCollect nebulizing spray device, making the sample preparation step more reproducible than manual spraying. Mass Spectrometry DESI-MS Flow rate: 1.5 μl/min Capillary voltage: 4-5kV Nebulising gas: 4-7 bar MALDI-MS Laser: Nd:YAG laser (355 nm) Pulse rate: 1000 Hz Data management The obtained data sets were subsequently processed using High Definition Imaging (HDI) software for detailed image analysis. On-tissue tryptic digestion analysed by MALDI A first example shows a rat brain tissue section analysed by MALDI imaging. First the tissue was washed with a series of ethanol based solution to remove salts present on the tissue, then a wash of chloroform to remove a large amount of lipids without delocalizing the protein. A trypsin solution was sprayed automatically using a commercial SunCollect nebulizing spray device, making the sample preparation step more reproducible than manual spraying prior to the matrix application, to cleave proteins into tryptic peptides. In figure 1,A) is demonstrated the type of ion images from either lipids and tryptic peptides that was obtained. Multimodal DESI and H&E for the analysing of clinical tissue. Consecutive tissue sections were analysed by DESI and MALDI imaging. Whereas MALDI imaging analysis is more of an established sort of analysis in the MSI, DESI has the main advantage of practically no sample preparation before the MSI analysis. Moreover by managing solvent and gas flow rates appropriately, in combination with the right voltages, the DESI technique results in negligible destruction of the tissue surface and therefore the same tissue sample can be histologically stained. In this case the molecular distribution was compared with the tissue's microscopic structure obtained from the H&E stained image. Figure 1. A) MALDI MSI ion images of lipids and tryptric peptides in a frozen rat brain tissue section. Figure 2. A) MS spectrum from the DESI imaging experiment in negative mode B) DESI Imaging Red & green overlay of m/z 771.52 (PG (36:3) - (red ion image)) and m/z 750.55 (PE (O-38:5) - (green ion image)) along with H&E image +overlays. CONCLUSION • On-tissue tryptic digested MALDI imaging experiment allowed the visualization of tryptic peptides. • DESI imaging of clinically relevant sample was overlaid with H&E stain image for morphological comparison. • DESI and MALDI imaging provides complementary rich information lipids allowing the tumour to healthy tissue type differentiation. Acknowledgements 1. This study was carried out in conjunction with Imperial College London. For the analysis of human samples, ethical approval was obtained from the National Research Ethics Service (NRES) Committee London – South East (Study ID 11/LO/0686). This work was supported by European Research Council under Starting Grant Scheme (Grant Agreement No: 210356) and the European Commission FP7 Intelligent Surgical Device project (contract no. 3054940). Figure 3. MALDI and DESI imaging comparison by PCA analysis vs healthy to tumour region on a human liver tissue section. C) PCA 1 loading plot. Analysis of a clinically relevant human liver sample 1 containing both healthy cells and a secondary tumor was performed. Figure 2,A) shows that endogenous lipid signals are plentiful from the DESI analysis. Image displayed in figure 2,B) is a Red/ Green ion images overlay with lipids m/z 771.52 PG (36:3)- (red ion image) and m/z 750.55 Phosphotidyl Ethanolamine (PE) (O-38:5) - (green ion image) specifically differentiating the tissue type. Figure 2, C), D), E) show the H&E stained image obtained from the same tissue and overlaid with the two lipid ion images from the DESI imaging experiment. Multimodal DESI and MALDI: complementarily information Regions of interest (ROIs) from both raw datasets were specified on the tumour and healthy part of the tissue and subjected to unsupervised multivariate analysis. Figure 3,A) shows the score PCA plot demonstrating the complementarity of the data obtained from DESI and MALDI imaging acquisition with a clear separation of their respective ROIs. Furthermore in 3,B) it is also possible to distinguish the ROIs extracted from the tumour type of the tissue vs. healthy. RESULTS
