Nouvelles stratégies analytiques favorisant l’augmentation de la spécificité et de la sensibilité en imagerie MS

Mass spectrometry (MS) is an analytical technique that measures the mass-to-charge ratio of ions. This technique is widely used in analytical chemistry to solve the molecular composition of complex homogenized samples. The use of homogenized samples means that all the information with respect to the initial distribution of analytes is lost. Imaging mass spectrometry (IMS) is an MS technique which is able to provide the spatial localization of a given analyte on a surface, such as thin tissue sections from various animal sources. One of the greatest advantages of IMS is its complementarity with histopathology which normally reveals the general structure of thin tissue sections as well as the localization of certain biomolecules such as proteins or of any molecules capable of specific interactions with an antibody. On the other hand, IMS is capable of imaging a wide variety of biomolecules ranging from small metabolites to the high molecular weight proteins and polymers. Among these, lipids are of particular interest for their key involvements in many biological processes. Their interest is even greater when considering that lipid imaging by classical histology is unable to differentiate between all lipid species. IMS of lipids is typically performed using matrix assisted laser desorption/ionization (MALDI). MALDI IMS can differentiate various classes of lipids and their localization within a thin tissue section by taking advantage of the specific affinity that different classes of lipids have for different matrices. The matrix deposition process, along with the choice of solvents, are key parameters that need to be considered in MALDI IMS. While MALDI offers great coverage of the phospholipidome, it fails miserably for neutral lipid analysis. Indeed, cholesterol and triacylglycerols (TAGs) are two classes of neutral lipids with very important vi biological roles which are extremely difficult to image by MALDI. We have developed three new strategies that enables the detection of neutral lipids, some of which are expressed in low abundance such as gangliosides, directly from thin tissue section using either MALDI or laser desorption/ionization (LDI). Cholesterol is a precursor of many key biomolecules such as vitamins and hormones. It’s also a major component of the cellular membrane. MALDI IMS allows in some cases imaging of the dehydrated form of cholesterol. Unfortunately, detecting cholesterol as such makes it impossible to distinguish some of its metabolites which ionize in a similar fashion and dissociate to produce the same ions. To address this issue, we have developed a new sample preparation method involving the deposition of a nanometer scale silver layer (16±2 nm) over a thin tissue section. This enables the detection by LDI MS of intact cholesterol and some fatty acid species as silver adducts with up to 5 µm in spatial resolution. Beyond cholesterol and fatty acids, TAGs is another class of highly abundant neutral lipids still poorly detected by MALDI IMS. As TAGs are the main molecules involved in energy storage of cells, they have been implicated in many metabolic diseases such as non- alcoholic fatty liver disease, atherosclerosis and even Alzheimer’s disease. The reason why TAGs are poorly detected by MALDI comes from two key factors. First, TAGs are unstable in acidic environments, typical of MALDI matrices. Second, competition effects for the ionizing proton provided by the MALDI matrix prevent TAGs from easily ionizing through this main ionization process. To overcome these limitations, we have developed a new two-step sample preparation method for TAG LDI IMS. We initially deposited a solution of carbonate buffer (pH 10.3, 85 mM) and sodium acetate (250 mM) on the tissue section to increase the amount vii of available sodium for enhanced TAG ionization. The second step consisted of sputtering a nanometer scale UV absorbing gold layer (28±3 nm) that allows for the detection of TAGs by LDI IMS with spatial resolution as low as 10 µm. When TAGs are present in low amounts in the tissue section, this method also enables the detection of cholesterol esters. Hunter’s disease is a genetic disease characterized by the abnormal accumulation of glucoaminoglycans (GAGs) and the secondary accumulation of gangliosides due to the lack of iduronate-2-sulfatase (IdS) enzyme which controls their degradation. The accumulation of both GAGs and gangliosides form deposits which induces various functional issues to different organs as well as neurologic disorders. To minimize these effects, an enzyme replacement therapy has been developed. Unfortunately, it shows efficacy in all organs except the brain due to the inability of the recombinant enzyme to cross the blood-brain barrier. To further our knowledge of the progression of the disease, using a mouse model of Hunter’s disease we have developed a MALDI based method to specifically image gangliosides in brain deposits with a spatial resolution of 5 µm. This method also permits subsequent ganglioside staining by immunohistochemtry of the tissue section. With this method, we have identified four types of ganglioside which are specific to the Hunter’s disease pathology. We were also able to detect two types of deposits, one which is enriched in short chain gangliosides and the other in long chain gangliosides.

Keyphrases: Sodium, Or, Acide gras, Imaging mass spectrometry, Silver, Gold, Fatty acid, Cholesterol ester, Hunter's disease