Molecular Mapping of In Situ CNS Lipids within EAE Tissue Using Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry




Kassim, Rawan Serena

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Multiple sclerosis (MS) is a neuroinflammatory disease that affects the central nervous system (CNS). A known pathological hallmark is lesions in the white matter of the brain and spinal cord. The lesions are a result of demyelination, the breakdown of myelin, which is a protective sheath made up of proteins and fatty substances. Myelin wraps around the nerve axons and allows for smooth electrical impulses throughout the CNS. Demyelinated axons have exposed fiber that disrupts nerve communication. The mechanism for demyelination has yet to be determined and the role that lipids play in the demyelination mechanism requires deeper exploration.

In the first study presented in this thesis, trapped ion mobility mass spectrometry time of flight (TIMS-TOF) with a matrix-assisted laser desorption/ionization (MALDI) source is used to create molecular mass maps of in situ lipids from experimental autoimmune encephalomyelitis (EAE) naïve mouse brain tissue. Matrices 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), and 9-aminoacridine (9-AA) are chosen to determine which lipids each matrix is successful in ionizing in both positive and negative mode. The mass spectrometry imaging (MSI) for mouse brain tissue shows that DHB is more successful in ionizing lipids in the positive mode. While DAN and 9-AA are more successful in the ionization of lipids in the negative mode. This could be because DHB is an acid that will readily give up a proton, and DAN and 9-AA are basic thus promoting deprotonation. The mass spectra show that DHB can ionize lipids of higher molecular weights, while DAN and 9-AA work better for lower molecular weight lipids. DHB and DAN were shown to be successful in mapping phospholipids, while 9-AA was the most successful in mapping sulfatides.

In the second study mouse spinal cord tissue was coated with matrix 9-AA. MSI was performed in negative mode ionization using MALDI-TOF. The MSI results show that the method used to map lipids in mouse brain tissue is unsuccessful for spinal cord tissue. MSI showed no localization of lipids on the tissue due to the analyte being washed to the edge of the tissue. Yet the mass spectra generated can be used to determine which lipids are present in the sample. Future work requires optimization of spinal cord tissue sample preparation to enhance ion signal localization on the tissue. Hematoxylin and eosin staining were used in both studies to confirm tissue histology. The staining was successful in confirming the histology of the tissue after collecting MSI data. The findings from both studies will be used to further investigate lipids in MS. Further analysis is required to confirm the role they play in the demyelination mechanism.



Analytical Chemistry, MALDI-TOF, Mass Spectrometry, Mass Spectrometry Imaging, Multiple Sclerosis, TIMS-TOF