| dc.description.abstract | Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for untargeted spatial investigation of complex biological systems, such as tissue sections. Confident analyte identification is challenging due to the high degree of mass redundancy, particularly in lipidomic analysis. Separation techniques, such as ion mobility spectrometry can be integrated with IMS workflows to help deconvolute this complexity. Here, trapped ion mobility spectrometry (TIMS) coupled with MALDI IMS was used to improve the specificity of a lipid IMS experiment, where a threefold increase in detected features was observed. This was achieved by resolving structurally distinct isobaric and isomeric interferences that overlap in the m/z dimension. Following this, a systematic analysis of common isomeric lipid standards, including sn-backbone, acyl chain, and double-bond position/stereochemistry isomers was demonstrated. As a proof-of-concept experiment, in-situ separation and visualization of lipid isomers with distinct spatial distributions was demonstrated in a whole-body mouse pup tissue section. These data provide the foundation for maximizing the sensitivity and specificity of lipid imaging experiments on TIMS-based MALDI platforms.
Building on these initial experiments, more complex analytes, such as gangliosides, were studied. Intact ganglioside analysis is challenging due to their high structural diversity and significant in-source fragmentation. Here, sample preparation strategies were optimized for in-situ MALDI IMS analysis of ganglioside species. Furthermore, we demonstrated the use of MALDI TIMS to distinguish disialylated isomers (GD1a and GD1b) in a 1:1 standard mixture, a total porcine brain ganglioside extract, and directly from murine tissue sections. With this, the unique spatial distributions of GD1a/b(d36:1) and GD1a/b(d38:1) isomers were revealed in murine nervous tissue samples at 20 µm spatial resolution.
The developed methods provided enhanced specificity necessary to analyze gangliosides within murine Staphylococcus aureus soft tissue abscesses. Ganglioside heterogeneity was qualitatively characterized in terms of glycan headgroup, ceramide chain, and sialic acid diversity. When considering TIMS-resolved species, isomeric monosialylated (GM1) species were also detected. Alteration of the relative isomer abundance (GM1b/GM1a) throughout the course of the infection was observed when analyzing 4, 6, and 10 days-post-infection tissue samples. Lastly, new molecular layers, unidentified by traditional histological assessment were revealed within the lesion. Ultimately, this work shows that MALDI TIMS IMS can be used as a highly informative technique to survey the molecular diversity within the host-pathogen interface. | |
