Development of a high-throughput shotgun-mass spectrometry method for qualitative and quantitative analysis of major mammalian brain gangliosides
The goal of this thesis was to develop a high-throughput shotgun-MS lipidomics method to qualitatively and quantitively analyze the major mammalian brain ganglioside classes: GM1, GD1, GT1 and GQ1. As a starting point for the method to be developed, a modified ganglioside extraction method from Svennerholm and Ladisch was used (Svennerholm and Fredman, 1980; Ladisch and Gillard, 1985). The efficiencies and the impact of different extraction procedures to the overall performance were evaluated with a software called OptiVal™. The evaluation showed that the most important steps of the protocol are the salt concentration of the water phase during the 2-phase extraction, and 10 mM NaCl yielded the best sensitivity. Also, the number of washing steps with water during reverse solid phase extraction using C18 resin has a significant effect. The next step was to find suitable standards for quantification of the individual ganglioside classes. Since deuterated and alike ganglioside standards were commercially not available, we initially used a deuterated PE standard with limited success. A collaboration with the Ludger Johannes lab provided us with modified C17-ganglioside standards. The term “modified” describes the enzymatic exchange of the fatty acid in the hydrophobic tail by a 17-carbon atom long fatty acid. Since odd numbered fatty acids occur very rarely in nature, it is possible to use the measured intensity of the modified ceramide headgroup of 35:1 (Sphingosine C18:1 + Fatty Acid C17:0) to quantify natural gangliosides. Ideally, we would need to have a fitting modified C17-ganglioside standard for each class to be quantified. Since first only GM1 as a modified standard was available, it was necessary to determine response factors (RFs) for the ganglioside classes GD1, GT1 and GQ1. RFs were assessed empirically by titrating a variety of equimolar concentrations of the modified C17-GM1 standard versus wildtype standards of the other ganglioside classes. After establishment of the RFs it was possible to determine the limits of detection (LOD) and quantification (LOQ) for the ganglioside classes GD1, GT1 and GQ1 - with regard to the modified C17-GM1 standard. When the modified C17-standards for GD1 and GT1 became available, I was able to find out whether the correct internal standards are superior to the proxy method via response factors. The results clearly showed that the use of a correct class standards is preferable. For GQ1 no modified C17-standard was obtainable, therefore this class still has to be quantified via RFs. Experiments showed that the modified C17-GT1 standard is best suited for that. Another major goal was to integrate the ganglioside method into the general lipid analysis workflow of the high-throughput shotgun mass spectrometry platform that we were using. To achieve these goals adjustments on the evaluated (=old) protocol had to be done. These adjustments included changes in the extraction steps from the Svennerholm & Ladisch more into the direction of a Bligh & Dyer based extraction method. This meant abandoning the 2-phase extraction step as well as the chloroform/methanol/water (C/M/W) 4:8:3 extraction, in favor of a C/M 10:1 followed by a C/M 2:1 extraction of 150 mM ammonium-bicarbonate water solution. The goal behind this was to enable a combination of the global lipidome extraction (Surma et al., 2015) with the ganglioside extraction. Another important improvement was scaling up the extraction process. The use of standard single solid phase extraction (SPE) cartridges was limiting the extraction throughput to only 24 samples at a time, therefore the single SPE cartridges were replaced with the 96-well SPE SOLA™ plates. To process the SOLA™ plates it was necessary to establish the usage of a vacuum manifold. Combined, these changes lowered the overall process time of the protocol from nearly two working days to one working day, without significant loss of sensitivity regarding the measured sample concentrations. This was assessed by performing the mouse brain tissue titration experiment, with all three modified C17-ganglioside class standards GM1, GD1 and GT1. Finally, the established method was applied to investigate the difference in ganglioside levels in the cerebellum compared to the brain hemispheres in mice of different age. First the C/M 10:1 and 2:1 extraction was done for the analysis of all non-ganglioside lipids in the sample. The leftover water phase was then loaded onto the SOLA™ plates and processed with the new protocol. The results matched the given goals - to establish a protocol to measure and quantify the four major brain ganglioside classes in combination with the global lipidomics in a high-throughput manner - and thus were a success. To the best of our knowledge, this was the first time such a broad lipidomic measurement has been performed, hence no other studies exist to which the outcome could be compared.
Ganglioside, Shotgun-MS, Mammalian Brain Gangliosides, Method Development, Gangliosid, Säugetiergehirnganglioside, Methodenentwicklung
Networked Digital Library of Theses and Dissertations