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Dissertation Submitted to the Combined Faculty of Natural Dissertation submitted to the Combined Faculty of Natural Sciences and Mathematics of the Ruperto Carola University Heidelberg, Germany for the degree of Doctor of Natural Sciences Presented by M.Sc. Johanna von Gerichten born in Speyer, Germany Oral examination: 13.11.201 Development of Mass Spectrometric Methods for Tissue Imaging and LC-based Quantification of Glycosphingolipids/Gangliosides including Tay-Sachs disease based Neuraminidase-deficient Mouse Models and Human Gut Microbiota Referees: Prof. Dr. Matthias Mayer Dr. PD Roger Sandhoff List of figures FIGURE 1: STRUCTURE OF CERAMIDE, SIMPLE AND COMPLEX GLYCOSPHINGOLIPIDS (GSL). .......................... 3 FIGURE 2: GLYCOSPHINGOLIPID BIOSYNTHESIS 4 WITH THE FOCUS ON GLUCOSYLCERAMIDE (GLCCER) BASED SYNTHESIS OF GANGLIOSIDES (LIGHT YELLOW). ................................................................................... 4 FIGURE 3: SUBCELLULAR COMPARTMENTALIZATION AND INTRACELLULAR TRAFFICKING OF HEXOSYLCERAMIDE AND GANGLIOSIDE BIOSYNTHESIS AND CATABOLISM 4. .......................................................................... 6 FIGURE 4: LYSOSOMAL GANGLIOSIDE CATABOLISM 4. ................................................................................. 11 FIGURE 5: SCAN MODES IN TANDEM MASS SPECTROMETRY. ....................................................................... 17 FIGURE 6: MASS SPECTROMETRY IMAGING (MSI) TECHNIQUES AND THEIR PRINCIPLE.. ............................... 19 FIGURE 7: TISSUE SAMPLING SCHEME OF MOUSE WT BL6 JEJUNUM, CAECUM AND COLON FOR THE ANALYSIS OF HEXOSYLCERAMIDES. .................................................................................................................. 23 FIGURE 8: IN-SOURCE DECAY (ISD) WITH LOSS OF MONOSACCHARIDES, AMMONIUM ADDUCT ION FORMATION, ISD WITH WATER LOSS, AND INFLUENCE OF SOURCE TEMPERATURE AND CAPILLARY VOLTAGE FOR THE GANGLIOSIDE STANDARDS GM3, GM2 AND GM1.) ............................................................................ 37 FIGURE 9: ADDUCT ION FORMATION IN THE PRESENCE OF 10 MM DIETHYLENETRIAMINE IN TOTAL GANGLIOSIDE PORCINE BRAIN STANDARD ............................................................................................................... 40 FIGURE 10: SEPARATION OF GANGLIOSIDES ON RP18 BASED CHROMATOGRAPHY (LEFT) AND HILIC BASED CHROMATOGRAPHY (RIGHT) OF NATURAL STANDARDS GM3, GM2 AND GM1. ..................................... 41 FIGURE 11: CALCULATED CHROMATOGRAPHIC RESOLUTION OF GGS GM1/GM3 FOR DIFFERENT SOLVENT SYSTEMS AND GRADIENTS USED FOR BEH C18 COLUMN.................................................................... 42 FIGURE 12: OVERLAY OF EXTRACTED ION CHROMATOGRAMS GENERATED BY HSS PFP COLUMN FOR GSL STANDARD MIX. ................................................................................................................................ 44 FIGURE 13: HILIC-MS2 METHOD DEVELOPMENT TESTING POSITIVE (A) AND NEGATIVE (B) ION MODE ........... 46 FIGURE 14: ADDITIVES TEST FOR GANGLIOSIDE DETECTION IN NEGATIVE ION MODE.. ................................... 47 FIGURE 15: SAMPLE PREPARATION TEST FOR THE ANALYSIS OF GANGLIOSIDES WITH HILIC-MS2 ANALYSIS.. 48 FIGURE 16: TLC OF GM2 GANGLIOSIDOSIS MOUSE BRAIN TISSUE AND NEURAMINIDASE 4 DEFICIENCY MOUSE BRAIN TISSUE FROM THE AGE OF 6 MONTH. ........................................................................................ 52 FIGURE 17: GANGLIOSIDE PATTERN IN MOUSE MODELS OF TAY-SACHS DISEASE (TSD) AND NEURAMINIDASE 4. ....................................................................................................................................................... 54 FIGURE 18: GANGLIOSIDE GM2 ACCUMULATION IN A MOUSE MODEL OF TAY-SACHS DISEASE COMBINED WITH NEURAMINIDASE 3 DEFICIENCY. ........................................................................................................ 55 FIGURE 19: DESI GEOMETRY AND DESI PARAMETER FOR OPTIMIZATION AND RHODAMINE SIGNAL. .............. 56 FIGURE 20: DESI PARAMETER OPTIMIZATION BY SPHINGOMYELIN STANDARD. ............................................. 57 FIGURE 21: DESI CHROMATOGRAM AND SPECTRUM FOR BRAIN TISSUE ACQUISITION IN FULL SCAN POSITIVE MODE AND PRECURSOR ION SCAN OF M/Z 184. .................................................................................. 57 I FIGURE 22: SAMPLE PREPARATION TEST FOR DESI-MS/MS SCANNING MOUSE WT CEREBELLUM WITH PRECURSOR ION SCAN FOR M/Z 184 AND DETECTING SPHINGOMYELIN AND PHOSPHATIDYLCHOLINE. .... 59 FIGURE 23: SAMPLE PREPARATION TEST FOR THE ON-TISSUE SAPONIFICATION OF PHOSPHOLIPIDS WITH 0.1 M KOH. .............................................................................................................................................. 60 FIGURE 24: DESI-MS/MS ANALYSIS OF WT MOUSE BRAIN LIPIDS IN NEGATIVE ION MODE WITH FULL SCAN (BOTTOM) AND PRECURSOR ION SCAN OF M/Z 241 FOR PHOSPHATIDYLINOSITOL (PI) AND PHOSPHATIDYLSERINE (PS) (TOP). ................................................................................................... 61 FIGURE 25: WORKFLOW DESI-MS/MS AND MALDI-TOF COMPARISON. ................................................... 62 FIGURE 26: DESI AND MALDI COMPARISON IN POSITIVE ION MODE USING FRESH FROZEN BRAIN SECTIONS IN COMBINATION WITH PRECURSOR ION MODE OF M/Z 184 FOR SPHINGOMYELIN (SM) AND PHOSPHATIDYLCHOLINE (PC) IN COMBINATION WITH WASH STEPS. ..................................................... 63 FIGURE 27: MALDI-TOF MSI OF GM1, GM2 AND GD1 IN SAGITTAL MOUSE BRAIN SLICES OF 3 MONTH OLD GM2 GANGLIOSIDOSIS AND NEU4 MODELS. ....................................................................................... 65 FIGURE 28: MALDI-TOF MSI OF GM1, GM2 AND GD1 IN SAGITTAL MOUSE BRAIN SLICES OF 6 MONTH OLD GM2 GANGLIOSIDOSIS, GD3S-/- AND NEU4-/- MODELS. ....................................................................... 66 FIGURE 29: MALDI-TOF MSI OF GM1, GM2 AND GA2 IN SAGITTAL MOUSE BRAIN SLICES OF 4.5 MONTH OLD GM2 GANGLIOSIDOSIS AND NEU3 MODELS.. ...................................................................................... 67 FIGURE 30: A: STEREOCHEMICAL STRUCTURES OF Β-GLCCER, Β-GALCER, AND Α-GALCER CONTAINING A C18-SPHINGOSINE AND AN N-LINKED PALMITIC ACID [HEXCER(D18:1/16:0)]. ...................................... 68 FIGURE 31: VARIOUS HEXOSYLCERAMIDE STANDARDS WERE SEPARATED ON A BORATE PRE-IMPREGNATED NORMAL PHASE HPTLC-PLATE AND VISUALIZED WITH ORCINOL REAGENT. .......................................... 69 FIGURE 32: A: HILIC SEPARATION OF A SYNTHETIC Α/Β-ANOMERIC MIXTURE OF GLCCER(D18:1/24:1) (DARK CYAN) AND A SYNTHETIC Α/Β-ANOMERIC MIXTURE OF GALCER(D18:1/24:1) (RED), EACH WITH APPROXIMATELY 15% Α-CONTENT. ................................................................................................... 71 FIGURE 33: RELATIVE DISTRIBUTION OF Β-GLCCER AND Β-GALCER IN VARIOUS MOUSE ORGANS. NS-, AS-, NP-, AND AP-HEXCERS WERE DETERMINED, WHICH CONTAINED A C18-SPHINGOID BASE AND N-BOUND FATTY ACIDS WITH THE CHAIN LENGTH C16 UP TO C26 (AS ANNOTATED)............................................. 72 FIGURE 34: HILIC-MS2-BASED QUANTIFICATION OF Β-GLCCERS AND Β- GALCERS IN LIVER OF WT, GBA2-/-, AND UGCGF/FALBCRE MICE .............................................................................................................. 73 FIGURE 35: HILIC-MS2-BASED SEPARATION OF AS-TYPE Β-GALCERS WITH 2R- AND 2S-HYDROXY STEARIC ACID. ............................................................................................................................................... 74 FIGURE 36: HILIC-MS2 BASED SEPARATION OF NS- AND AS-TYPE Β-GLCCER AND Β-GALCER IN MOUSE BRAIN LIPID EXTRACTS ENRICHED IN NEUTRAL GSLS OF WT, GM2 GANGLIOSIDOSIS AND NEURAMINIDASE DEFICIENCY. .................................................................................................................................... 76 FIGURE 37: HILIC-MS2-BASED DETECTION OF Α-GALCERS FROM BACTEROIDES FRAGILIS AND IDENTIFICATION OF EQUIVALENT COMPOUNDS IN BACTEROIDES VULGATUS AND PREVOTELLA COPRI. ........................... 77 FIGURE 38: HILIC-MS2 BASED ANALYSIS OF WT BL6 AND AXENIC NMRI MOUSE INTESTINAL TRACT. ........... 78 II FIGURE 39: HILIC-MS2 BASED ANALYSIS OF WT BL6 MOUSE INTESTINAL TRACT COMPARTMENTS JEJUNUM, CAECUM AND COLON OF Β-GLCCER (A), Β-GALCER (B) AND PREDICTED BDS-Α-GALCER (C). ............. 79 FIGURE 40: HILIC-MS2 BASED ANALYSIS OF HEXOSYLCERAMIDES IN LIPID EXTRACTS FROM CAECUM OF MICE FED FOR 5 DAYS TWICE A DAY WITH EITHER 100 µL PBS, OLIVE OIL, OR MILK FAT (GHEE) IN ADDITION TO CHOW DIET OR HIGH FAT DIET. .......................................................................................................... 80 III List of tables TABLE 1: INSTRUMENTS FOR SAMPLE PREPARATION .................................................................................. 20 TABLE 2: INSTRUMENTS FOR TLC ANALYSIS. ............................................................................................. 20 TABLE 3: LC ANALYTICAL COLUMNS FROM WATERS GMBH (ESCHBORN, GERMANY). .................................. 21 TABLE 4: INSTRUMENTS FOR IMAGING MASS SPECTROMETRY. ................................................................... 21 TABLE 5: MOUSE STRAINS FOR THE ANALYSIS OF TAY-SACHS DISEASE AND THE INVOLVEMENT OF NEURAMINIDASES. ............................................................................................................................ 22 TABLE 6: BACTERIA STRAINS USED FOR HILIC-MS2 ANALYSIS. .................................................................. 23 TABLE 7: SOLVENTS AND ADDITIVES
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