Subcellular metabolite and lipid analysis of \u3ci\u3eXenopus laevis\u3c/i\u3e eggs by LAESI mass spectrometry

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis

Matrix Assisted Laser Desorption Ionization (MALDI) Mass Spectrometry: An Imaging Modality to Monitor the Effects of Gene Therapy in a Murine Model of GM1 Gangliosidosis

GM1 gangliosidosis is an autosomal recessive lysosomal storage disorder caused by an enzyme deficiency of β-galactosidase (β-gal) leading to toxic accumulation of GM1 ganglioside in the central nervous system (CNS) and progressive neurodegeneration. Adeno-associated virus (AAV) mediated gene delivery of lysosomal enzymes to the CNS has shown great potential for the treatment of lysosomal storage diseases with neurological involvement. In this work we use MALDI mass spectrometry imaging (MSI) to assess the spatial distribution of gangliosides, ganglioside metabolites and related lipids in a GM1 gangliosidosis mouse brain model following adeno-associated virus (AAV) gene therapy. Deficiency of β-galactosidase enzyme in a β-gal-/- mouse brain showed an overall 8-fold increase in GM1 relative to the control by MSI analysis, with specific spatial localization based on its ceramide content. Bilateral thalamic injection of AAVrh10-bgal in β-gal-/- mice significantly reduced GM1 levels relative to untreated β-gal-/- mice. The therapeutic efficacy of this approach is through distribution of functional enzyme via axonal transport through the extensive connectivity of the thalamus with most of the brain, with some exceptions such as the temporal cortex. Accordingly MSI showed AAV gene therapy reduced GM1 nearly to the control levels in all regions of the brain except in the temporal cerebral cortex. This correlated with low levels of bgal in this brain region as assessed by histochemical staining of tissue sections. MSI also detected asialo-GM1 and other ganglioside metabolites elevated in untreated β-gal-/- mice, which were also reduced after AAV therapy. Interestingly sulfated galactocereberosides reduced in the myelin sheath in untreated β-gal-/- mice were restored to normal levels after AAV therapy. Overall, this study demonstrates that MALDI MSI can be used to map specific target analytes and their metabolites while also offering the ability to detect unanticipated effects caused by gene therapy

Direct Detection of Diverse Metabolic Changes in Virally Transformed and Tax-Expressing Cells by Mass Spectrometry

International audiencen.

Single cell analysis and tissue imaging by laser ablation and mass spectrometry

The direct analysis of biochemicals in tissues and single cells is critical for understanding living organisms. Due to excellent selectivity and sensitivity, mass spectrometry (MS) has proven to be an invaluable tool for the analysis of the biomolecules. Recent developments in atmospheric pressure direct ionization sources have enabled the in situ analysis of biological samples without external influences (e.g., purification, extraction, matrix addition etc.) that might alter their biochemical makeup. The work presented in this dissertation shows my efforts to utilize two novel atmospheric pressure (AP) direct ionization methodologies, AP infrared (IR) matrix-assisted laser desorption ionization (MALDI) and laser ablation electrospray ionization (LAESI) MS, for metabolomics, tissue imaging and single cell analysis. Chapter 1 introduces analytical techniques used for the analysis of tissues and single cells. The fundamental aspects of IR laser ablation and its utilization in two direct ionization techniques, AP IR-MALDI and LAESI, are reviewed. Chapter 2 introduces AP IR-MALDI for MS. It presents proof-of-principle molecular imaging of mock peptide samples at atmospheric pressure. The utility of AP IR-MALDI for plant tissue imaging and metabolomics are also discussed. Chapter 3 describes the AP IR-MALDI analysis of various pharmaceuticals directly in their commercial formulations, as well as endogenous metabolites, exogenous drug metabolites and synthetic polymers in urine. Chapter 4 presents the application of mid-infrared laser ablation for molecular imaging. The dynamics of the ablation plume and ion production in AP IR-MALDI and LAESI are compared. In Chapter 5 metabolites and lipids are identified in mouse brain sections using MS with AP IR-MALDI and LAESI ion production. Reactive LAESI relies on interactions between the laser ablated species and reactants, e.g., Li +, introduced through the electrospray. This new modality of LAESI enables the analysis of samples with otherwise low ion yields. Chapter 6 discusses the metabolic analysis of single cells by MS at atmospheric pressure. This breakthrough is made possible by the tight focusing of mid-IR laser light through an etched optical fiber tip and accurate aiming of cells for ablation through visualization and micromanipulation. Similar to conventional LAESI, the ablated plume is postionized by an electrospray. Chapter 7 surveys the major challenges in the field of atmospheric pressure ion production based on mid-IR laser ablation. The need for the analysis of smaller cells, reactive LAESI-MS, ultrahigh resolution LAESI-MS, and the potential application of LAESI-MS in laser surgery are discussed

Ablation and analysis of small cell populations and single cells by consecutive laser pulses

Abstract Laser ablation of single cells through a sharpened optical fiber is used for the detection of metabolites by laser ablation electrospray ionization (LAESI) mass spectrometry (MS). Ablation of the same Allium cepa epidermal cell by consecutive pulses indicates the rupture of the cell wall by the second shot. Intracellular sucrose heterogeneity is detected by subsequent laser pulses pointing to rupturing the vacuolar membrane by the third exposure. Ion production by bursts of laser pulses shows that the drying of ruptured A. cepa cells occurs in ∼50 s at low pulse rates (10 pulses/s bursts) and significantly faster at high pulse rates (100 pulses/s bursts). These results point to the competing role of cytoplasm ejection and evaporative drying in diminishing the LAESI-MS signal in ∼50 s or 100 laser pulses, whichever occurs first

Redox phospholipidomics analysis reveals specific oxidized phospholipids and regions in the diabetic mouse kidney

While it is generally accepted that oxidative stress impacts the diabetic kidney and contributes to pathogenesis, there is a substantial lack of knowledge about the molecular entity and anatomic location of a variety of reactive species. Here we provide a novel “oxidative stress map” of the diabetic kidney – the first of its kind, and identify specific, oxidized and other reactive lipids and their location. We used the db/db mouse model and Desorption Electrospray Ionization (DESI) mass spectrometry combined with heatmap image analysis. We analyzed a comprehensive array of phospholipid peroxide species in normal (db/m) and diabetic (db/db) kidneys using DESI imaging. Oxilipidomics heatmaps of the kidneys were generated focusing on phospholipids and their potential peroxidized products. We identified those lipids that undergo peroxidation in diabetic nephropathy. Several phospholipid peroxides and their spatial distribution were identified that were specific to the diabetic kidney, with significant enrichment in oxygenated phosphatidylethanolamines (PE) and lysophosphatidylethanolamine. Beyond qualitative and semi-quantitative information about the targets, the approach also reveals the anatomic location and the extent of lipid peroxide signal propagation across the kidney. Our approach provides novel, in-depth information of the location and molecular entity of reactive lipids in an organ with a very heterogeneous landscape. Many of these reactive lipids have been previously linked to programmed cell death mechanisms. Thus, the findings may be relevant to understand what impact phospholipid peroxidation has on cell and mitochondria membrane integrity and redox lipid signaling in diabetic nephropathy