Correlated Heterospectral Lipidomics for Biomolecular Profiling of Remyelination in Multiple Sclerosis

Analyzing lipid composition and distribution within the brain is important to study white matter pathologies that present focal demyelination lesions, such as multiple sclerosis. Some lesions can endogenously re-form myelin sheaths. Therapies aim to enhance this repair process in order to reduce neurodegeneration and disability progression in patients. In this context, a lipidomic analysis providing both precise molecular classification and well-defined localization is crucial to detect changes in myelin lipid content. Here we develop a correlated heterospectral lipidomic (HSL) approach based on coregistered Raman spectroscopy, desorption electrospray ionization mass spectrometry (DESI-MS), and immunofluorescence imaging. We employ HSL to study the structural and compositional lipid profile of demyelination and remyelination in an induced focal demyelination mouse model and in multiple sclerosis lesions from patients ex vivo. Pixelwise coregistration of Raman spectroscopy and DESI-MS imaging generated a heterospectral map used to interrelate biomolecular structure and composition of myelin. Multivariate regression analysis enabled Raman-based assessment of highly specific lipid subtypes in complex tissue for the first time. This method revealed the temporal dynamics of remyelination and provided the first indication that newly formed myelin has a different lipid composition compared to normal myelin. HSL enables detailed molecular myelin characterization that can substantially improve upon the current understanding of remyelination in multiple sclerosis and provides a strategy to assess remyelination treatments in animal models

Development of robust DESI imaging MS instrumentation for analysis of tissue samples

Desorption electrospray ionisation (DESI) is an ambient ionisation method that can be used for mass spectrometric imaging. Due to its ability to ionise lipids, its non-destructive nature and its minimal sample preparation, it is particularly suitable for biological tissue imaging and it can be combined directly with classical histopathological staining methods. However, despite having been one of the earliest ambient ionisation techniques, first published more than ten years ago, DESI still suffers from repeatability and reproducibility issues. The aim of this project was to identify and eliminate the primary sources of variability in DESI. One major source of variability was found to be solvent capillary positioning in the DESI sprayer. The ideal positioning of the capillary was hypothesised to be perfect centering, although this could not be achieved in practice. However, a fixed capillary position as close to the central position as possible was successfully implemented. This eliminated movement and vibration of the capillary, improving repeatability. By using a tapered, machine-cut, small inner diameter capillary the operational parameters could be optimised for improved spatial resolution. The improved sprayer was combined with a fast-scanning QToF MS for fast, high spatial resolution DESI-MS imaging. Tests on rat brain sections showed that DESI was able to distinguish between different tissue types even with a more than tenfold increase of scan speed. The improved DESI source was also used to analyse mouse and human brain tissue sections as part of a larger study on remyelination in multiple sclerosis. It was shown that DESI can be combined with Raman spectroscopy to provide complementary imaging information, although the two methods could not be performed on the same sections. An alternative, closely related ambient ionisation method, desorption electro-flow focusing ionisation (DEFFI) was tested for tissue imaging performance and repeatability. DEFFI uses a co-flowing gas stream to focus a charged solvent into a jet, making the primary spray inherently concentric. Repeatability was similar to a carefully optimised DESI sprayer and after adjustment of operational parameters, its imaging resolution was comparable. The comparison of DESI and DEFFI data suggested that the data was sufficiently similar to allow integration of DEFFI into existing DESI workflows. Finally, the impact of MS inlet capillary dimensions and heating was investigated. These experiments suggested that ion production in DESI partially occurs in the inlet capillary. A small capillary inner diameter was found to be crucial for dissociation of ion clusters. Capillary heating was shown to improve overall sensitivity and also to make DESI less sensitive to geometrical changes. This supports the hypothesis that some desolvation and ion formation occurs during droplet transfer into the MS. Overall, the work presented here brings DESIMS imaging closer to becoming a routine tool in clinical diagnostics.Open Acces

Simultaneous determination of polycyclic aromatic hydrocarbons and their chlorination by-products in drinking water and the coatings of water pipes by automated solid-phase microextraction followed by gas chromatography–mass spectrometry

International audienceIn this study, an automated method for the simultaneous determination of polycyclic aromatic hydrocarbons (PAHs) and their chlorination by-products in drinking water was developed based on online solid-phase microextraction-gas chromatography–mass spectrometry. The main focus was the optimisation of the solid-phase microextraction step. The influence of the agitation rate, type of fibre, desorption time, extraction time, extraction temperature, desorption temperature, and solvent addition was examined. The method was developed and validated using a mixture of 17 PAHs, 11 potential chlorination by-products (chlorinated and oxidised PAHs) and 6 deuterated standards. The limit of quantification was 10 ng/L for all target compounds. The validated method was used to analyse drinking water samples from three different drinking water distribution networks and the presumably coal tar-based pipe coatings of two pipe sections. A number of PAHs were detected in all three networks although individual compositions varied. Several PAH chlorination by-products (anthraquinone, fluorenone, cyclopenta[d,e,f]phenanthrenone, 3-chlorofluoranthene, and 1-chloropyrene) were also found, their presence correlating closely with that of their respective parent compounds. Their concentrations were always below 100 ng/L. In the coatings, all PAHs targeted were detected although concentrations varied between the two coatings (76–12,635 mg/kg and 12–6295 mg/kg, respectively). A number of chlorination by-products (anthraquinone, fluorenone, cyclopenta[d,e,f]phenanthrenone, 3-chlorofluoranthene, and 1-chloropyrene) were also detected (from 40 to 985 mg/kg), suggesting that the reaction of PAHs with disinfectant agents takes place in the coatings and not in the water phase after migration

High resolution ambient MS imaging of biological samples by desorption electro-flow focussing ionization

In this study, we examine the suitability of desorption electro-flow focusing ionization (DEFFI) for mass spectrometry imaging (MSI) of biological tissue. We also compare the performance of desorption electrospray ionization (DESI) with and without the flow focusing setup. The main potential advantages of applying the flow focusing mechanism in DESI is its rotationally symmetric electrospray jet, higher intensity, more controllable parameters, and better portability due to the robustness of the sprayer. The parameters for DEFFI have therefore been thoroughly optimized, primarily for spatial resolution but also for intensity. Once the parameters have been optimized, DEFFI produces similar images to the existing DESI. MS images for mouse brain samples, acquired at a nominal pixel size of 50 μm, are comparable for both DESI setups, albeit the new sprayer design yields better sensitivity. Furthermore, the two methods are compared with regard to spectral intensity as well as the area of the desorbed crater on rhodamine-coated slides. Overall, the implementation of a flow focusing mechanism in DESI is shown to be highly suitable for imaging biological tissue and has potential to overcome some of the shortcomings experienced with the current geometrical design of DESI

Research data supporting "Correlated heterospectral lipidomics for biomolecular profiling of remyelination in multiple sclerosis"

Research data supporting the paper: Bergholt, M.S. et al., "Correlated heterospectral lipidomics for biomolecular profiling of remyelination in multiple sclerosis", ACS Central Science, 2017, DOI: 10.1021/acscentsci.7b00367

Investigation of the Impact of Desorption Electrospray Ionization Sprayer Geometry on Its Performance in Imaging of Biological Tissue

In this study, the impact of sprayer design and geometry on performance in desorption electrospray ionization mass spectrometry (DESI-MS) is assessed, as the sprayer is thought to be a major source of variability. Absolute intensity repeatability, spectral composition, and classification accuracy for biological tissues are considered. Marked differences in tissue analysis performance are seen between the commercially available and a lab-built sprayer. These are thought to be associated with the geometry of the solvent capillary and the resulting shape of the primary electrospray. Experiments with a sprayer with a fixed solvent capillary position show that capillary orientation has a crucial impact on tissue complex lipid signal and can lead to an almost complete loss of signal. Absolute intensity repeatability is compared for five lab-built sprayers using pork liver sections. Repeatability ranges from 1 to 224% for individual sprayers and peaks of different spectral abundance. Between sprayers, repeatability is 16%, 9%, 23%, and 34% for high, medium, low, and very low abundance peaks, respectively. To assess the impact of sprayer variability on tissue classification using multivariate statistical tools, nine human colorectal adenocarcinoma sections are analyzed with three lab-built sprayers, and classification accuracy for adenocarcinoma versus the surrounding stroma is assessed. It ranges from 80.7 to 94.5% between the three sprayers and is 86.5% overall. The presented results confirm that the sprayer setup needs to be closely controlled to obtain reliable data, and a new sprayer setup with a fixed solvent capillary geometry should be developed