| 1. |
- Lanekoff, Ingela, 1975, et al.
(författare)
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An in situ fracture device to image lipids in single cells using ToF-SIMS
- 2011
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Ingår i: SURFACE AND INTERFACE ANALYSIS. - : Wiley. - 0142-2421 .- 1096-9918. ; 43:1-2, s. 257-260
-
Tidskriftsartikel (refereegranskat)abstract
- Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) imaging yields molecule-specific images showing the spatial distribution of specific lipids with submicron resolution, making it a potentially powerful method for studying a variety of biological questions in single cells. In order to make possible the analysis of hydrated cells in vacuum, we have designed a device for in situ freeze-fracture of cell samples. PC12 cells are frozen between two silicon shards in a sandwich geometry, and the fracture is conducted under vacuum inside the analysis chamber of the instrument. After freeze-fracture, ToF-SIMS analysis was carried out using a Bi cluster ion source, providing high-resolution images of single cells. We also show that when combining freeze-fracture and cluster SIMS imaging, low abundant biologically important lipids, specifically PE, PC and non-SM PC, can be imaged in single cells. Finally, K+ ion localization was used as a diagnostic to identify fracture planes through the cell for these fractured samples.
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| 2. |
- Mossberg, Ann-Kristin, et al.
(författare)
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HAMLET Interacts with Lipid Membranes and Perturbs Their Structure and Integrity.
- 2010
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 5:2
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Cell membrane interactions rely on lipid bilayer constituents and molecules inserted within the membrane, including specific receptors. HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a tumoricidal complex of partially unfolded alpha-lactalbumin (HLA) and oleic acid that is internalized by tumor cells, suggesting that interactions with the phospholipid bilayer and/or specific receptors may be essential for the tumoricidal effect. This study examined whether HAMLET interacts with artificial membranes and alters membrane structure. METHODOLOGY/PRINCIPAL FINDINGS: We show by surface plasmon resonance that HAMLET binds with high affinity to surface adherent, unilamellar vesicles of lipids with varying acyl chain composition and net charge. Fluorescence imaging revealed that HAMLET accumulates in membranes of vesicles and perturbs their structure, resulting in increased membrane fluidity. Furthermore, HAMLET disrupted membrane integrity at neutral pH and physiological conditions, as shown by fluorophore leakage experiments. These effects did not occur with either native HLA or a constitutively unfolded Cys-Ala HLA mutant (rHLA(all-Ala)). HAMLET also bound to plasma membrane vesicles formed from intact tumor cells, with accumulation in certain membrane areas, but the complex was not internalized by these vesicles or by the synthetic membrane vesicles. CONCLUSIONS/SIGNIFICANCE: The results illustrate the difference in membrane affinity between the fatty acid bound and fatty acid free forms of partially unfolded HLA and suggest that HAMLET engages membranes by a mechanism requiring both the protein and the fatty acid. Furthermore, HAMLET binding alters the morphology of the membrane and compromises its integrity, suggesting that membrane perturbation could be an initial step in inducing cell death.
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| 3. |
- Ali, Ahmed, et al.
(författare)
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Single cell metabolism : current and future trends
- 2022
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Ingår i: Metabolomics. - : Springer. - 1573-3882 .- 1573-3890. ; 18:10
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Forskningsöversikt (refereegranskat)abstract
- Single cell metabolomics is an emerging and rapidly developing field that complements developments in single cell analysis by genomics and proteomics. Major goals include mapping and quantifying the metabolome in sufficient detail to provide useful information about cellular function in highly heterogeneous systems such as tissue, ultimately with spatial resolution at the individual cell level. The chemical diversity and dynamic range of metabolites poses particular challenges for detection, identification and quantification. In this review we discuss both significant technical issues of measurement and interpretation, and progress toward addressing them, with recent examples from diverse biological systems. We provide a framework for further directions aimed at improving workflow and robustness so that such analyses may become commonly applied, especially in combination with metabolic imaging and single cell transcriptomics and proteomics.
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| 4. |
- Arora, Abishek, et al.
(författare)
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Screening autism-associated environmental factors in differentiating human neural progenitors with fractional factorial design-based transcriptomics
- 2023
-
Ingår i: Scientific Reports. - : Springer Nature. - 2045-2322. ; 13
-
Tidskriftsartikel (refereegranskat)abstract
- Research continues to identify genetic variation, environmental exposures, and their mixtures underlying different diseases and conditions. There is a need for screening methods to understand the molecular outcomes of such factors. Here, we investigate a highly efficient and multiplexable, fractional factorial experimental design (FFED) to study six environmental factors (lead, valproic acid, bisphenol A, ethanol, fluoxetine hydrochloride and zinc deficiency) and four human induced pluripotent stem cell line derived differentiating human neural progenitors. We showcase the FFED coupled with RNA-sequencing to identify the effects of low-grade exposures to these environmental factors and analyse the results in the context of autism spectrum disorder (ASD). We performed this after 5-day exposures on differentiating human neural progenitors accompanied by a layered analytical approach and detected several convergent and divergent, gene and pathway level responses. We revealed significant upregulation of pathways related to synaptic function and lipid metabolism following lead and fluoxetine exposure, respectively. Moreover, fluoxetine exposure elevated several fatty acids when validated using mass spectrometry-based metabolomics. Our study demonstrates that the FFED can be used for multiplexed transcriptomic analyses to detect relevant pathway-level changes in human neural development caused by low-grade environmental risk factors. Future studies will require multiple cell lines with different genetic backgrounds for characterising the effects of environmental exposures in ASD.
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| 5. |
- Bemis, Kylie A., et al.
(författare)
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Statistical detection of differentially abundant ions in mass spectrometry-based imaging experiments with complex designs
- 2019
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Ingår i: International Journal of Mass Spectrometry. - : ELSEVIER SCIENCE BV. - 1387-3806 .- 1873-2798. ; 437, s. 49-57
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Tidskriftsartikel (refereegranskat)abstract
- Mass Spectrometry Imaging (MSI) characterizes changes in chemical composition between regions of biological samples such as tissues. One goal of statistical analysis of MSI experiments is class comparison, i.e. determining analytes that change in abundance between conditions more systematically than as expected by random variation. To reach accurate and reproducible conclusions, statistical analysis must appropriately reflect the initial research question, the design of the MSI experiment, and all the associated sources of variation. This manuscript highlights the importance of following these general statistical principles. Using the example of two case studies with complex experimental designs, and with different strategies of data acquisition, we demonstrate the extent to which choices made at key points of this workflow impact the results, and provide suggestions for appropriate design and analysis of MSI experiments that aim at detecting differentially abundant analytes. (C) 2018 Elsevier B.V. All rights reserved.
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| 6. |
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| 7. |
- Bergman, Hilde-Marlene, et al.
(författare)
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Metabolite aberrations in early diabetes detected in rat kidney using mass spectrometry imaging
- 2019
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer Science and Business Media LLC. - 1618-2642 .- 1618-2650. ; 411:13, s. 2809-2816
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Tidskriftsartikel (refereegranskat)abstract
- Diabetic kidney disease is a serious complication of diabetes that can ultimately lead to end-stage renal disease. The pathogenesis of diabetic kidney disease is complex, and fundamental research is still required to provide a better understanding of the driving forces behind it. We report regional metabolic aberrations from an untargeted mass spectrometry imaging study of kidney tissue using an insulinopenic rat model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidneys were harvested 2weeks thereafter. Imaging was performed using nanospray desorption electrospray ionization connected to a high-mass-resolving mass spectrometer. No histopathological changes were observed in the kidney sections; however, mass spectrometry imaging revealed a significant increase in several 18-carbon unsaturated non-esterified fatty acid species and monoacylglycerols. Notably, these 18-carbon acyl chains were also constituents of several increased diacylglycerol species. In addition, a number of short- and long-chain acylcarnitines were found to be accumulated while several amino acids were depleted. This study presents unique regional metabolic data indicating a dysregulated energy metabolism in renal mitochondria as an early response to streptozotocin-induced type I diabetes.
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| 8. |
- Bergman, Hilde-Marléne, et al.
(författare)
-
Quantitative mass spectrometry imaging of small-molecule neurotransmitters in rat brain tissue sections using nanospray desorption electrospray ionization
- 2016
-
Ingår i: The Analyst. - : Royal Society of Chemistry (RSC). - 0003-2654 .- 1364-5528. ; 141:12, s. 3686-3695
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Tidskriftsartikel (refereegranskat)abstract
- Small molecule neurotransmitters are essential for the function of the nervous system, and neurotransmitter imbalances are often connected to neurological disorders. The ability to quantify such imbalances is important to provide insights into the biochemical mechanisms underlying the disorder. This proof-of-principle study presents online quantification of small molecule neurotransmitters, specifically acetylcholine, γ-aminobutyric acid (GABA) and glutamate, in rat brain tissue sections using nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging. By incorporating deuterated internal standards in the nano-DESI solvent we show identification, accurate mapping, and quantification of these small neurotransmitters in rat brain tissue without introducing any additional sample preparation steps. We find that GABA is about twice as abundant in the medial septum-diagonal band complex (MSDB) as in the cortex, while glutamate is about twice as abundant in the cortex as compared to the MSDB. The study shows that nano-DESI is well suited for imaging of small molecule neurotransmitters in health and disease.
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| 9. |
- Bergman, Hilde-Marlene, et al.
(författare)
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Single‐Cell Mass Spectrometry
- 2018
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Ingår i: Encyclopedia of Analytical Chemistry. - Chichester, UK : Wiley-VCH Verlagsgesellschaft. - 9780470027318
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Bokkapitel (refereegranskat)abstract
- Over the past few decades, the chemical characterization of single cells has improved immensely. In particular, mass spectrometry (MS) has pioneered direct analysis of metabolites, lipids, and peptides from single cells. This progress has been enabled by new and improved strategies for ionization and sampling, where a multitude of techniques for single‐cell MS has contributed unique insights to many different disciplines. Here, an overview of the main three techniques secondary ion mass spectrometry (SIMS), matrix‐assisted laser desorption ionization (MALDI), and ambient ionization for direct single‐cell MS analysis are presented, including some example studies detailing the use of single‐cell MS.
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| 10. |
- Bui Quang, Dong, et al.
(författare)
-
Simultaneous determination of four organic acids in beverages by capillary electrophoresis coupled with ultraviolet detector
- 2022
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Ingår i: Vietnam Journal of Food Control. - : National Institute for Food Control. - 2615-9252. ; 5:2, s. 89-103
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Tidskriftsartikel (refereegranskat)abstract
- A simple and rapid capillary electrophoresis method with direct ultraviolet (UV) detection was set up for the determination of four organic acids in beverages. The method included dilution and filtration as simple sample preparation steps. The electrophoretic separation and detection of oxalic, malic, citric and lactic acids in wines and beers were performed in 8 min. For the method validation, linearity, detection and quantification limits, repeatability and recovery in wine and beer matrices were studied. Good linearity was observed from 25 to 500 mg/L for all acids excluding lactic acid, for which it started from 50 mg/L. The limits of quantitation of oxalic, malonic and citric acid were set 9.5 to 28.5 mg/L. Repeatability of this method was from 3.2 to 7.3%, recoveries ranged from 90.1 to 110.1%. The validated method was applied to the analysis of different wines and beers and showed great variability in their composition.
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| 11. |
- Cardoso-Palacios, Carlos, et al.
(författare)
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Direct Analysis of Pharmaceutical Drugs Using Nano-DESI MS
- 2016
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Ingår i: Journal of Analytical Methods in Chemistry. - : Hindawi Limited. - 2090-8865 .- 2090-8873.
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Tidskriftsartikel (refereegranskat)abstract
- Counterfeit pharmaceutical drugs imply an increasing threat to the global public health. It is necessary to have systems to control the products that reach the market and to detect falsified medicines. In this work, molecules in several pharmaceutical tablets were directly analyzed using nanospray desorption electrospray ionization mass spectrometry (nano-DESI MS). Nano-DESI is an ambient surface sampling technique which enables sampling of molecules directly from the surface of the tablets without any sample pretreatment. Both the active pharmaceutical ingredients (APIs) and some excipients were detected in all analyzed tablets. Principal component analysis was used to analyze mass spectral features from different tablets showing strong clustering between tablets with different APIs. The obtained results suggest nano-DESI MS as future tool for forensic analysis to discern APIs present in unknown tablet samples.
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| 12. |
- Carter, Sarah-Sophia, 1994-, et al.
(författare)
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PDMS leaching and its implications for on-chip studies focusing on bone regeneration applications
- 2020
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Ingår i: Organs-on-a-Chip. - : Elsevier. - 2666-1020. ; 2
-
Tidskriftsartikel (refereegranskat)abstract
- Polydimethylsiloxane (PDMS) is among the most widely used materials for organ-on-chip systems. Despite itsmultiple beneficial characteristics from an engineering point of view, there is a concern about the effect of PDMSon the cells cultured in such devices. The aim of this study was to enhance the understanding of the effect of PDMSon cellular behavior in a context relevant for on-chip studies. The focus was put on an indirect effect of PDMS,namely leaching of uncrosslinked oligomers, particularly for bone regeneration applications. PDMS-based chipswere prepared and analyzed for the potential release of PDMS oligomers within the microfluidic channel whenkept at different flow rates. Leaching of uncrosslinked oligomers from PDMS was quantified as silicon concen-tration by inductively coupled plasma - optical emission spectrometry and further confirmed by mass spec-trometry. Subsequently, PDMS-leached media, with a silicon concentration matching the on-chip experiment,were prepared to study cell proliferation and osteogenic differentiation of MC3T3-E1 pre-osteoblasts and humanmesenchymal stem cells. The silicon concentration initially detected in the media was inversely proportional tothe tested flow rates and decreased to control levels within 52 h. In addition, by curing the material overnightinstead of 2 h, regardless of the curing temperature (65 and 80 C), a large reduction in silicon concentration wasfound, indicating the importance of the PDMS curing parameters. Furthermore, it was shown that PDMS oligo-mers enhanced the differentiation of MC3T3-E1 pre-osteoblasts, this being a cell type dependent effect as nochanges in cell differentiation were observed for human mesenchymal stem cells. Overall, this study illustrates theimportance of optimization steps when using PDMS devices for biological studies, in particular PDMS curingconditions and extensive washing steps prior to an experiment.
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| 13. |
- Duncan, Kyle D., et al.
(författare)
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Advances in mass spectrometry based single-cell metabolomics
- 2019
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Ingår i: The Analyst. - : ROYAL SOC CHEMISTRY. - 0003-2654 .- 1364-5528. ; 144:3, s. 782-793
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Forskningsöversikt (refereegranskat)abstract
- Metabolomics has grown into a prominent field contributing to the molecular understanding of complex biological processes in both health and disease. Furthermore, single-cells are known to display metabolic differences between seemingly homogeneous populations of cells. Single-cell metabolomics attempts to analyze many cellular metabolites from single cells to understand phenotypic heterogeneity, which is a significant challenge due to the low analyte abundances and limited sample volumes. Label-free metabolite detection can be achieved with mass spectrometry, which is capable of simultaneously analyzing hundreds of metabolites. Herein, we review the recent advances in mass spectrometry based single-cell metabolomics, highlighting the current state-of-the-art within the last three years, and identify the challenges to move the field forward.
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| 14. |
- Duncan, Kyle D., et al.
(författare)
-
In situ imaging reveals disparity between prostaglandin localization and abundance of prostaglandin synthases
- 2021
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 4:1
-
Tidskriftsartikel (refereegranskat)abstract
- Duncan et al. use a mass spectrometry imaging method to assess the localization and concentration of prostaglandins (PGs) in mouse tissues during pregnancy. This study brings new biological insights into the spatial evaluation of PGs in tissues, which could reveal the functional significance of each PGs during different stages of embryo development/pregnancy. Prostaglandins are important lipids involved in mediating many physiological processes, such as allergic responses, inflammation, and pregnancy. However, technical limitations of in-situ prostaglandin detection in tissue have led researchers to infer prostaglandin tissue distributions from localization of regulatory synthases, such as COX1 and COX2. Herein, we apply a novel mass spectrometry imaging method for direct in situ tissue localization of prostaglandins, and combine it with techniques for protein expression and RNA localization. We report that prostaglandin D-2, its precursors, and downstream synthases co-localize with the highest expression of COX1, and not COX2. Further, we study tissue with a conditional deletion of transformation-related protein 53 where pregnancy success is low and confirm that PG levels are altered, although localization is conserved. Our studies reveal that the abundance of COX and prostaglandin D-2 synthases in cellular regions does not mirror the regional abundance of prostaglandins. Thus, we deduce that prostaglandins tissue localization and abundance may not be inferred by COX or prostaglandin synthases in uterine tissue, and must be resolved by an in situ prostaglandin imaging.
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| 15. |
- Duncan, Kyle D., et al.
(författare)
-
Membrane Sampling Separates Naphthenic Acids from Biogenic Dissolved Organic Matter for Direct Analysis by Mass Spectrometry
- 2022
-
Ingår i: Environmental Science and Technology. - : American Chemical Society (ACS). - 0013-936X .- 1520-5851. ; 56:5, s. 3096-3105
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Tidskriftsartikel (refereegranskat)abstract
- Oil sands process waters can release toxic naphthenic acids (NAs) into aquatic environments. Analytical techniques for NAs are challenged by sample complexity and interference from naturally occurring dissolved organic matter (DOM). Herein, we report the use of a poly(dimethylsiloxane) (PDMS) polymer membrane for the on-line separation of NAs from DOM and use direct infusion electrospray ionization mass spectrometry to yield meaningful qualitative and quantitative information with minimal sample cleanup. We compare the composition of membrane-permeable species from natural waters fortified with a commercial NA mixture to those derived from weak anion exchange solid-phase extraction (SPE) using high-resolution mass spectrometry. The results show that SPE retains a wide range of carboxylic acids, including biogenic DOM, while permeation through PDMS was selective for petrogenic classically defined NAs (CnH2n+zO2). A series of model compounds (log K-ow similar to 1-7) were used to characterize the perm-selectivity and reveal the separation is based on hydrophobicity. This convenient sample cleanup method is selective for the O-2 class of NAs and can be used prior to conventional analysis or as an on-line analytical strategy when coupled directly to mass spectrometry.
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| 16. |
- Duncan, Kyle D., et al.
(författare)
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Spatially Defined Surface Sampling Capillary Electrophoresis Mass Spectrometry
- 2019
-
Ingår i: Analytical Chemistry. - : American Institute of Chemical Engineers. - 0003-2700 .- 1520-6882. ; 91:12, s. 7819-7827
-
Tidskriftsartikel (refereegranskat)abstract
- Capillary electrophoresis mass spectrometry (CE-MS) is an established technique for targeted and untargeted analysis of metabolites from complex biological samples. However, current CE-MS devices rely on liquid sample extracts, which restricts acquisition of spatially defined chemical information from tissue samples. The ability to chemically profile distinct cellular regions in tissue can contribute better understanding to molecular foundations in health and disease. Therefore, we describe the first CE-MS device capable of untargeted metabolite profiling directly from defined morphological regions of solid tissue sections. With surface sampling capillary electrophoresis mass spectrometry (SS-CE-MS), endogenous molecules are sampled and detected from a single defined tissue location. Characterization of SS-CE MS from different locations of the outer epidermal layer of A. Cepa demonstrated reproducible relative migration times and a peak area RSD of 20% (n = 5). Further, relative migration times were conserved for endogenous metabolites in tissues with varying complexities, including brain, spinal cord, and kidney. Results from proof-of-principle experiments from distinct morphological tissue regions reveal simultaneous analysis of small and large biomolecules, confident metabolite annotation, identification of in-source fragmentation interferences, and discrete isomeric abundances related to biological function. We envision that this new tool will provide in-depth chemical profiling and annotation of molecules in distinct cellular regions of tissue for improved biological understanding.
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| 17. |
- Golubova, Anastasia, 1996-, et al.
(författare)
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Surface sampling capillary electrophoresis–mass spectrometry for a direct chemical characterization of tissue and blood samples
- 2023
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Ingår i: Electrophoresis. - : John Wiley & Sons. - 0173-0835 .- 1522-2683. ; 44:3-4, s. 387-394
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Tidskriftsartikel (refereegranskat)abstract
- Capillary electrophoresis (CE) is a powerful separation tool for non-targeted analysis of chemically complex samples, such as blood, urine, and tissue. However, traditionally CE requires samples in solution for analysis, which limits information on analyte distribution and heterogeneity in tissue. The recent development of surface sampling CE–mass spectrometry (SS-CE–MS) brings these advantages of CE to solid samples and enables chemical mapping directly from the tissue surface without laborious sample preparation. Here, we describe developments of SS-CE–MS to increase reproducibility and stability for metabolite, lipid, and protein extraction from tissue sections and dried blood spots. Additionally, we report the first electrokinetic sequential sample injection for high throughput analysis. We foresee that the wide molecular coverage from a distinct tissue region in combination with higher throughput will provide novel information on biological function and dysfunction.
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| 18. |
- Lanekoff, Ingela, 1975
(författare)
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Analysis of phospholipids in cellular membranes with LC and imaging mass spectrometry
- 2011
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Imaging mass spectrometry enables the creation of molecule specific images from the surface of a solid sample in vacuum. To solve the issue of bringing single cells into vacuum without altering their native distribution of molecules, a freeze fracture device that fits the time of flight secondary ion mass spectrometry (TOF-SIMS) IV instrument has been developed. This makes it possible to get a snapshot of the chemical distribution across frozen hydrated single cells that are only 10-20 µm in diameter. The cells of interest in this thesis are rat pheochromocytoma (PC12) cells. PC12 cells resemble and act like neurons in the sense that upon stimulation they release dopamine, which is a substance used for communication between neurons. In previous studies using these model cells, the rate of this release has been shown to change after the cells have been incubated with different phospholipids. To investigate the amount of phospholipids that have accumulated in the plasma membrane of PC12 cells after an overnight incubation, the combination of the freeze fracture device and the TOF-SIMS IV instrument was utilized. Relative to the endogenous phospholipid the results show that 0.5% of phosphatidylcholine (PC) and 1.3% of phosphatidylethanolamine (PE) had accumulated in the plasma membrane. Together with previous results on changes in the release of dopamine in PC12 cells, this suggests that the phospholipid composition of the plasma membrane of neurons is highly regulated. This gives a hint as to the importance of phospholipids during this highly important cellular process. The technique of liquid chromatography (LC) mass spectrometry (MS) does not provide molecular information in images but has the ability to separate similar molecules in a sample. This is of high importance when analyzing a specific molecule in a complex sample. Anaerobic ammonium oxidizing (anammox) bacteria reside in sediment on the ocean floor. These bacteria are highly important to the environment because they convert biologically available nitrogen into dinitrogen gas (N2), which is returned to the atmosphere. By denitrifying biologically available nitrogen they limit the risk of over fertilization in the ocean. They are also believed to contribute greatly to the global N2 production. By combining LCMS with an extensive sample clean up procedure a phospholipid biomarker for viable anammox bacteria has been used to detect the location of anammox bacteria in a sediment core sample.
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| 19. |
- Lanekoff, Ingela, Assoc. Prof. 1975-, et al.
(författare)
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Imaging of lipids and metabolites using nanospray desorption electrospray ionization mass spectrometry.
- 2015
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Ingår i: Methods in molecular biology. - New York, NY : Springer. - 9781493913565 ; , s. 99-106
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Bokkapitel (refereegranskat)abstract
- Nanospray desorption electrospray ionization (nano-DESI) is an ambient ionization technique that uses localized liquid extraction for mass spectrometry imaging of molecules on surfaces. Nano-DESI enables imaging of ionizable molecules from a sample in its native state without any special sample pretreatment. In this chapter we describe the protocol for nano-DESI imaging of thin tissue sections.
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| 20. |
- Lanekoff, Ingela, 1975, et al.
(författare)
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Mass spectrometry imaging of freeze-dried membrane phospholipids of dividing Tetrahymena pyriformis
- 2013
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Ingår i: Surface and Interface Analysis. - : Wiley. - 0142-2421 .- 1096-9918. ; 45:1, s. 211-214
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Tidskriftsartikel (refereegranskat)abstract
- Time of Flight secondary ion mass spectrometry (TOF-SIMS) has been used to explore the distribution of phospholipids in the plasma membrane of Tetrahymena pyriformis during cell division. The dividing cells were freeze-dried prior to analysis followed by line scan and region of interest analysis at various stages of cell division. The results showed no signs of phospholipid domain formation at the junction between the dividing cells. Instead the results showed that the sample preparation technique had a great impact on one of the examined phospholipids, namely phosphatidylcholine (PC). Phosphatidylcholine and 2-aminoethylphosphonolipid (2-AEP) have therefore been evaluated in Tetrahymena cells that have been subjected to different sample preparation techniques: freeze drying ex situ, freeze fracture, and freeze fracture with partial or total freeze drying in situ. The result suggests that freeze drying ex situ causes the celia to collapse and cover the plasma membrane.
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| 21. |
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| 22. |
- Lanekoff, Ingela, 1975, et al.
(författare)
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Relative quantification of phospholipid accumulation in the PC12 cell plasma membrane following phospholipid incubation using TOF-SIMS Imaging.
- 2011
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 83:13, s. 5337-5343
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Tidskriftsartikel (refereegranskat)abstract
- Time of flight secondary ion mass spectrometry (TOF-SIMS) imaging has been used to investigate the incorporation of phospholipids into the plasma membrane of PC12 cells after incubation with phosphatidylcholine (PC) and phosphatidylethanolamine (PE). The incubations were done at concentrations previously shown to change the rate of exocytosis in model cell lines. The use of TOF-SIMS in combination with an in situ freeze fracture device enables the acquisition of ion images from the plasma membrane in single PC12 cells. By incubating cells with deuterated phospholipids and acquiring ion images at high mass resolution, specific deuterated fragment ions were used to monitor the incorporation of lipids into the plasma membrane. The concentration of incorporated phospholipids relative to the original concentration of PC was thus determined. The observed relative amounts of phospholipid accumulation in the membrane ranges from 0.5 to 2 percent following 19 hours of incubation with PC at 100 to 300 μM and from 1 to 9 percent following incubation with PE at the same concentrations. Phospholipid accumulation is therefore shown to be dependent on the concentration in the surrounding media. In combination with previous exocytosis results, the present data suggests that very small changes in the plasma membrane phospholipid concentration are sufficient to produce significant effects on important cellular processes, such as exocytosis in PC12 cells.
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| 23. |
- Lanekoff, Ingela, Prof. 1975-, et al.
(författare)
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Single-cell metabolomics : where are we and where are we going?
- 2022
-
Ingår i: Current Opinion in Biotechnology. - : Elsevier. - 0958-1669 .- 1879-0429. ; 75
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Forskningsöversikt (refereegranskat)abstract
- Single-cell metabolomics with mass spectrometry enables a large variety of metabolites to be simultaneously detected from individual cells, without any preselection or labelling, to map phenotypes on the single cell level. Although the field is relatively young, it is steadily progressing with an increasing number of active research groups, techniques for cell sampling and ionization, tools for data analysis, and applications to answer important biomedical and environmental questions. In addition, the community shows great creativity in overcoming challenges associated with low sample volumes, a wide range of metabolite species, and large datasets. Here, we briefly discuss publications since 2019 and aim to provide the unfamiliar reader with an insight into the field and the expert reader with an update on the current status of the field.
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| 24. |
- Lanekoff, Ingela, 1975, et al.
(författare)
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Time of Flight Mass Spectrometry Imaging of Samples Fractured In Situ with a Spring-Loaded Trap System.
- 2010
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Ingår i: Analytical chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 82:15, s. 6652-6659
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Tidskriftsartikel (refereegranskat)abstract
- An in situ freeze fracture device featuring a spring-loaded trap system has been designed and characterized for time of flight secondary ion mass spectrometry (TOF SIMS) analysis of single cells. The device employs the sandwich assembly, which is typically used in freeze fracture TOF SIMS experiments to prepare frozen, hydrated cells for high-resolution SIMS imaging. The addition of the spring-loaded trap system to the sandwich assembly offers two advances to this sample preparation method. First, mechanizing the fracture by adding a spring standardizes each fracture by removing the need to manually remove the top of the sandwich assembly with a cryogenically cooled knife. A second advance is brought about because the top of the sandwich is not discarded after the sandwich assembly has been fractured. This results in two imaging surfaces effectively doubling the sample size and providing the unique ability to image both sections of a cell bifurcated by the fracture. Here, we report TOF SIMS analysis of freeze fractured rat pheochromocytoma (PC12) cells using a Bi cluster ion source. This work exhibits the ability to obtain single cell chemical images with subcellular lateral resolution from cells preserved in an ice matrix. In addition to preserving the cells, the signal from lipid fragment ions rarely identified in single cells are better observed in the freeze-fractured samples for these experiments. Furthermore, using the accepted argument that K(+) signal indicates a cell that has been fractured though the cytoplasm, we have also identified different fracture planes of cells over the surface. Coupling a mechanized freeze fracture device to high-resolution cluster SIMS imaging will provide the sensitivity and resolution as well as the number of trials required to carry out biologically relevant SIMS experiments.
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| 25. |
- Lanekoff, Ingela, Assoc. Prof. 1975-, et al.
(författare)
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Trp53 deficient mice predisposed to preterm birth display region-specific lipid alterations at the embryo implantation site
- 2016
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Ingår i: Scientific Reports. - : Springer Science and Business Media LLC. - 2045-2322. ; 6
-
Tidskriftsartikel (refereegranskat)abstract
- Here we demonstrate that conditional deletion of mouse uterine Trp53 (p53(d/d)), molecularly linked to mTORC1 activation and causally linked to premature uterine senescence and preterm birth, results in aberrant lipid signatures within the heterogeneous cell types of embryo implantation sites on day 8 of pregnancy. In situ nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) was used to characterize the molecular speciation of free fatty acids, monoacylglycerol species, unmodified and oxidized phosphatidylcholine (PC/Ox-PC), and diacylglycerol (DG) species within implantation sites of p53(d/d) mice and floxed littermates. Implantation sites from p53(d/d) mice exhibited distinct spatially resolved changes demonstrating accumulation of DG species, depletion of Ox-PC species, and increase in species with more unsaturated acyl chains, including arachidonic and docosahexaenoic acid. Understanding abnormal changes in the abundance and localization of individual lipid species early in the progression to premature birth is an important step toward discovering novel targets for treatments and diagnosis.
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| 26. |
- Lillja, Johan, et al.
(författare)
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Correlative image analysis for in-depth chemical analysis using multiple parallelized mass spectrometry imaging with high resolution and MSn
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Attaining specificity in mass spectrometry imaging (MSI) is challenging and often results in longer experimental runtime or lower spatial resolution. Herein we describe a novel method for acquisition of MS2 spectra in an ion trap parallel with high resolution accurate mass FTMS scans. Using the nanospray desorption electrospray ionization (nano-DESI) ion source for MSI, we were able to perform 28 MS2 events in an ion trap simultaneously to recording a transient for FTMS. This allowed for deep characterization of the targeted compounds and allowed for improved annotation by spatially correlating the FTMS and ITMS2 data. By combining the data streams from data with different specificities we were able to efficiently data mine the complex data set and could annotate several lipid species.
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| 27. |
- Lillja, Johan, et al.
(författare)
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Determination of Monounsaturated Fatty Acid Isomers in Biological Systems by Modeling MS3 Product Ion Patterns
- 2020
-
Ingår i: Journal of the American Society for Mass Spectrometry. - WASHINGTON DC USA : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 31:12, s. 2479-2487
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Tidskriftsartikel (refereegranskat)abstract
- Unsaturated free fatty acids are natively present in biological samples as isomers, where double bonds can be situated on different carbons in the acyl chain. While these isomers can have different actions and impacts on biological systems, they are inherently difficult to identify and differentiate by mass spectrometry alone. To address this challenge, several techniques for derivatization of the double bond or metal cationization at the carboxylic group have yielded diagnostic product ions for the respective isomer in tandem mass spectrometry. However, diagnostic product ions do not necessarily reflect quantitative isomeric ratios since fatty acid isomers have different ionization and fragmentation efficiencies. Here, we introduce a simple and rapid approach to predict the quantitative ratio of isomeric monounsaturated fatty acids. Specifically, empirically derived MS3 product ion patterns from fatty acid silver adducts are modeled using a stepwise linear model. This model is then applied to predict the proportion oleic and vaccenic acid in chemically complex samples at individual concentrations between 0.45 and 5.25 mu M, with an average accuracy and precision below 2 and 5 mol %, respectively. We show that by simply including silver ions in the electrospray solvent, isomeric ratios are rapidly predicted in neat standards, rodent plasma, and tissue extract. Furthermore, we use the method to directly map isomeric ratios in tissue sections using nanospray desorption electrospray ionization MS3 imaging without any sample preparation or modification to the instrumental setup. Ultimately, this approach provides a simple and rapid solution to differentiate monounsaturated fatty acids using commonly available commercial mass spectrometers without any instrumental modifications.
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| 28. |
- Lillja, Johan, et al.
(författare)
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Ion-to-Image, i2i, a Mass Spectrometry Imaging Data Analysis Platform for Continuous Ionization Techniques
- 2023
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:31, s. 11589-11595
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry imaging (MSI) techniques generate data that reveal spatial distributions of molecules on a surface with high sensitivity and selectivity. However, processing large volumes of mass spectrometry data into useful ion images is not trivial. Furthermore, data from MSI techniques using continuous ionization sources where data are acquired in line scans require different data handling strategies compared to data collected from pulsed ionization sources where data are acquired in grids. In addition, for continuous ionization sources, the pixel dimensions are influenced by the mass spectrometer duty cycle, which, in turn, can be controlled by the automatic gain control (AGC) for each spectrum (pixel). Currently, there is a lack of data-handling software for MSI data generated with continuous ionization sources and AGC. Here, we present ion-to-image (i2i), which is a MATLAB-based application for MSI data acquired with continuous ionization sources, AGC, high resolution, and one or several scan filters. The source code and a compiled installer are available at https://github.com/LanekoffLab/i2i. The application includes both quantitative, targeted, and nontargeted data processing strategies and enables complex data sets to be processed in minutes. The i2i application has high flexibility for generating, processing, and exporting MSI data both from simple full scans and more complex scan functions interlacing MSn and SIM scan data sets, and we anticipate that it will become a valuable addition to the existing MSI software toolbox.
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| 29. |
- Lillja, Johan
(författare)
-
Novel strategies to increase throughput and differentiate lipid isomers in mass spectrometry imaging : Development of computational tools and complex mass spectrometric methods for nanospray desorption electrospray ionization
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In this thesis, method development for improved analyte identification and throughput in mass spectrometry imaging (MSI) is discussed. In MSI, the spatial distribution of analytes from a sample is determined and visualized, information about the detected molecules interaction within the sample can thereby the deduced. Most MSI methods utilize high resolution accurate mass (HRAM) to assign an identity to a feature by its mass-to-charge (m/z) value. However, HRAM cannot distinguish isomeric species. I have therefore developed novel tools for annotation and separation of lipid isomer for MSI with nanospray desorption electrospray ionization (nano-DESI). Specifically, I show that tandem mass spectrometry (MSn) of silver ion species of lipids can be used for the separation of both fatty acid and phospholipid isomers. Additionally, I developed a method for parallelized MSn experiments, by performing multiple ion trap MSn in parallel to a fourier transform mass spectrometry (FTMS) transient. The ion trap MSn, albeit with lower resolution, has orthogonal specificity to FTMS and therefore generates a data set where the analytes identity can be deduced. Because the ITMS is executed in parallel to the typically used FTMS scan the imaging parameters are kept constant, thus generating a richer data set without increasing spatial resolution or experimental runtime.Lastly, data sets generated with nano-DESI MSI are complex and require specialized software tools for processing. I also discuss an open-source tool for data processing with high flexibility and fast processing speeds. With the newly developed tool we were able to process and interrogate data sets, thereby making better use of the acquired data.
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| 30. |
- Lillja, Johan, et al.
(författare)
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Quantitative determination of sn-positional phospholipid isomers in MSn using silver cationization
- 2022
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 414:25, s. 7473-7482
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Tidskriftsartikel (refereegranskat)abstract
- Glycerophospholipids are one of the fundamental building blocks for life. The acyl chain connectivity to the glycerol backbone constitutes different sn-positional isomers, which have great diversity and importance for biological function. However, to fully realize their impact on function, analytical techniques that can identify and quantify sn-positional isomers in chemically complex biological samples are needed. Here, we utilize silver ion cationization in combination with tandem mass spectrometry (MSn) to identify sn-positional isomers of phosphatidylcholine (PC) species. In particular, a labile carbocation is generated through a neutral loss (NL) of AgH, the dissociation of which provides diagnostic product ions that correspond to acyl chains at the sn-1 or sn-2 position. The method is comparable to currently available methods, has a sensitivity in the nM-mu M range, and is compatible with quantitative imaging using mass spectrometry in MS4. The results reveal a large difference in isomer concentrations and the ion images show that the sn-positional isomers PC 18:1_18:0 are homogeneously distributed, whereas PC 18:1_16:0 and PC 20:1_16:0 show distinct localizations to sub-hippocampal structures.
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| 31. |
- Lindfors, Lina
(författare)
-
Molecular Imaging of Diabetic Kidney Tissue and Binding Studies of Proinsulin C-peptide
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Diabetic kidney disease is a serious complication of diabetes with a complex and incompletely understood pathology. In this work, the molecular changes in diabetic rat kidneys at a very early disease stage were studied using nanospray desorption electrospray ionisation mass spectrometry imaging. Our results demonstrate how disease-relevant metabolites and lipids can be conveniently analysed on intact kidney tissue sections. A number of significantly increased metabolites were identified in the diabetic kidney, revealing disturbances in energy metabolism detectable before histological changes.Proinsulin C-peptide is produced in the pancreas along with insulin and has shown beneficial effects in diabetes, but its mode of action is not yet known. 125I radiolabelled C-peptide was used to study its tissue distribution in healthy and diabetic rats after intravenous injection. The majority of C-peptide accumulated in renal tissues, with lower levels in the diabetic animals, showing that there are significant changes in kidney – C-peptide interactions in early stage diabetes.The interactions of C-peptide with the orphan receptor GPR146, which has been proposed as its receptor, were also investigated using Chinese hamster ovary cells overexpressing human GPR146. Neither dynamic mass redistribution nor β-arrestin recruitment assays showed any significant response to human or murine C-peptides in the GPR146 overexpressing cells compared to controls. Fluorescence confocal microscopy revealed no surface binding or cellular uptake of C-peptides by GPR146 overexpressing cells compared to controls. These combined results refute the suggestion that GPR146 is the C-peptide receptor.To further probe the function of C-peptide, 15N-labelled residues were incorporated into the peptide in preparation for nanoscale secondary ion mass spectrometry imaging of cells and intact kidney tissue sections. A number of crosslinking C-peptides were also designed and synthesised for experiments aimed at identifying its binding target. These studies have not yet been completed. Finally, to investigate the structure-activity relationship of C-peptide, a library of modified pentapeptide analogues was created for medium-throughput testing in a cell assay.
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| 32. |
- Marques, Cátia, et al.
(författare)
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A Direct Infusion Probe for Rapid Metabolomics of Low-Volume Samples
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:37, s. 12875-12883
-
Tidskriftsartikel (refereegranskat)abstract
- Targeted and nontargeted metabolomics has the potential to evaluate and detect global metabolite changes in biological systems. Direct infusion mass spectrometric analysis enables detection of all ionizable small molecules, thus simultaneously providing information on both metabolites and lipids in chemically complex samples. However, to unravel the heterogeneity of the metabolic status of cells in culture and tissue a low number of cells per sample should be analyzed with high sensitivity, which requires low sample volumes. Here, we present the design and characterization of the direct infusion probe, DIP. The DIP is simple to build and position directly in front of a mass spectrometer for rapid metabolomics of chemically complex biological samples using pneumatically assisted electrospray ionization at 1 mu L/min flow rate. The resulting data is acquired in a square wave profile with minimal carryover between samples that enhances throughput and enables several minutes of uniform MS signal from 5 mu L sample volumes. The DIP was applied to study the intracellular metabolism of insulin secreting INS-1 cells and the results show that exposure to 20 mM glucose for 15 min significantly alters the abundance of several small metabolites, amino acids, and lipids.
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| 33. |
- Marques-Santos, Cátia M., et al.
(författare)
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Global and Spatial Metabolomics of Individual Cells Using a Tapered Pneumatically Assisted nano-DESI Probe
- 2023
-
Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 34:11, s. 2518-2524
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Tidskriftsartikel (refereegranskat)abstract
- Single-cell metabolomics has the potential to reveal unique insights into intracellular mechanisms and biological processes. However, the detection of metabolites from individual cells is challenging due to their versatile chemical properties and concentrations. Here, we demonstrate a tapered probe for pneumatically assisted nanospray desorption electrospray ionization (PA nano-DESI) mass spectrometry that enables both chemical imaging of larger cells and global metabolomics of smaller 15 mu m cells. Additionally, by depositing cells in predefined arrays, we show successful metabolomics from three individual INS-1 cells per minute, which enabled the acquisition of data from 479 individual cells. Several cells were used to optimize analytical conditions, and 93 or 97 cells were used to monitor metabolome alterations in INS-1 cells after exposure to a low or high glucose concentration, respectively. Our analytical approach offers insights into cellular heterogeneity and provides valuable information about cellular processes and responses in individual cells.
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| 34. |
- Mavroudakis, Leonidas, 1994-, et al.
(författare)
-
CpG preconditioning reduces accumulation of lysophosphatidylcholine in ischemic brain tissue after middle cerebral artery occlusion
- 2021
-
Ingår i: Analytical and Bioanalytical Chemistry. - : Springer. - 1618-2642 .- 1618-2650. ; 413, s. 2735-2745
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Tidskriftsartikel (refereegranskat)abstract
- Ischemic stroke is one of the major causes of death and permanent disability in the world. However, the molecular mechanisms surrounding tissue damage are complex and further studies are needed to gain insights necessary for development of treatment. Prophylactic treatment by administration of cytosine-guanine (CpG) oligodeoxynucleotides has been shown to provide neuroprotection against anticipated ischemic injury. CpG binds to Toll-like receptor 9 (TLR9) causing initialization of an inflammatory response that limits visible ischemic damages upon subsequent stroke. Here, we use nanospray desorption electrospray ionization (nano-DESI) mass spectrometry imaging (MSI) to characterize molecular effects of CpG preconditioning prior to middle cerebral artery occlusion (MCAO) and reperfusion. By doping the nano-DESI solvent with appropriate internal standards, we can study and compare distributions of phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) in the ischemic hemisphere of the brain despite the large changes in alkali metal abundances. Our results show that CpG preconditioning not only reduces the infarct size but it also decreases the degradation of PC and accumulation of LPC species, which indicates reduced cell membrane breakdown and overall ischemic damage. Our findings show that molecular mechanisms of PC degradation are intact despite CpG preconditioning but that these are limited due to the initialized inflammatory response.
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| 35. |
- Mavroudakis, Leonidas, 1994-, et al.
(författare)
-
Host-Guest Chemistry for Simultaneous Imaging of Endogenous Alkali Metals and Metabolites with Mass Spectrometry
- 2022
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:5, s. 2391-2398
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Tidskriftsartikel (refereegranskat)abstract
- Sodium and potassium are biological alkali metal ions that are essential for the physiological processes of cells and organisms. In combination with small-molecule metabolite information, disturbances in sodium and potassium tissue distributions can provide a further understanding of the biological processes in diseases. However, methods using mass spectrometry are generally tailored toward either elemental or molecular detection, which limits simultaneous quantitative mass spectrometry imaging of alkali metal ions and molecular ions. Here, we provide a new method by including crown ether molecules in the solvent for nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) that combines host-guest chemistry targeting sodium and potassium ions and quantitative imaging of endogenous lipids and metabolites. After evaluation and optimization, the method was applied to an ischemic stroke model, which has highly dynamic tissue sodium and potassium concentrations, and we report 2 times relative increase in the detected sodium concentration in the ischemic region compared to healthy tissue. Further, in the same experiment, we showed the accumulation and depletion of lipids, neurotransmitters, and amino acids using relative quantitation with internal standards spiked in the nano-DESI solvent. Overall, we demonstrate a new method that with a simple modification in liquid extraction MSI techniques using hostguest chemistry provides the added dimension of alkali metal ion imaging to provide unique insights into biological processes.
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| 36. |
- Mavroudakis, Leonidas, 1994-, et al.
(författare)
-
Identification and Imaging of Prostaglandin Isomers Utilizing MS3 Product Ions and Silver Cationization
- 2023
-
Ingår i: Journal of the American Society for Mass Spectrometry. - : American Chemical Society (ACS). - 1044-0305 .- 1879-1123. ; 34:10, s. 2341-2349
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Tidskriftsartikel (refereegranskat)abstract
- Prostaglandins (PGs) are important lipid mediators involved in physiological processes, such as inflammation and pregnancy. The pleiotropic effects of the PG isomers and their differential expression from cell types impose the necessity for studying individual isomers locally in tissue to understand the molecular mechanisms. Currently, mass spectrometry (MS)-based analytical workflows for determining the PG isomers typically require homogenization of the sample and a separation method, which results in a loss of spatial information. Here, we describe a method exploiting the cationization of PGs with silver ions for enhanced sensitivity and tandem MS to distinguish the biologically relevant PG isomers PGE2, PGD2, and Δ12-PGD2. The developed method utilizes characteristic product ions in MS3 for training prediction models and is compatible with direct infusion approaches. We discuss insights into the fragmentation pathways of Ag+ cationized PGs during collision-induced dissociation and demonstrate the high accuracy and robustness of the model to predict isomeric compositions of PGs. The developed method is applied to mass spectrometry imaging (MSI) of mouse uterus implantation sites using silver-doped pneumatically assisted nanospray desorption electrospray ionization and indicates localization to the antimesometrial pole and the luminal epithelium of all isomers with different abundances. Overall, we demonstrate, for the first time, isomeric imaging of major PG isomers with a simple method that is compatible with liquid-based extraction MSI methods.
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| 37. |
- Mavroudakis, Leonidas, 1994-, et al.
(författare)
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Ischemic Stroke Causes Disruptions in the Carnitine Shuttle System
- 2023
-
Ingår i: Metabolites. - : MDPI. - 2218-1989 .- 2218-1989. ; 13:2
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Tidskriftsartikel (refereegranskat)abstract
- Gaining a deep understanding of the molecular mechanisms underlying ischemic stroke is necessary to develop treatment alternatives. Ischemic stroke is known to cause a cellular energy imbalance when glucose supply is deprived, enhancing the role for energy production via β-oxidation where acylcarnitines are essential for the transportation of fatty acids into the mitochondria. Although traditional bulk analysis methods enable sensitive detection of acylcarnitines, they do not provide information on their abundances in various tissue regions. However, with quantitative mass spectrometry imaging the detected concentrations and spatial distributions of endogenous molecules can be readily obtained in an unbiased way. Here, we use pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging (PA nano-DESI MSI) doped with internal standards to study the distributions of acylcarnitines in mouse brain affected by stroke. The internal standards enable quantitative imaging and annotation of endogenous acylcarnitines is achieved by studying fragmentation patterns. We report a significant accumulation of long-chain acylcarnitines due to ischemia in brain tissue of the middle cerebral artery occlusion (MCAO) stroke model. Further, we estimate activities of carnitine transporting enzymes and demonstrate disruptions in the carnitine shuttle system that affects the β-oxidation in the mitochondria. Our results show the importance for quantitative monitoring of metabolite distributions in distinct tissue regions to understand cell compensation mechanisms involved in handling damage caused by stroke.
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| 38. |
- Piwowar, A. M., et al.
(författare)
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C60-ToF SIMS imaging of frozen hydrated HeLa cells
- 2013
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Ingår i: Surface and Interface Analysis. - : Wiley. - 0142-2421 .- 1096-9918. ; 45:1, s. 302-304
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Tidskriftsartikel (refereegranskat)abstract
- Sample preparation continues to be a major challenge for SIMS studies of biological materials. Maintaining the native hydrated state of the material is important for preserving both chemical and spatial information. Here, we discuss a method that combines a sample wash and dry protocol followed by plunge-freezing in liquid ethane for a frozen-hydrated analysis of mammalian cells (HeLa). This method allows for the removal of the growth medium and maintains the hydrated state of the cells so that they can be prepared frozen-hydrated without the need for a freeze-fracture device. The cells, which were grown on silicon, were successfully regrown after the cleaning procedure, confirming that a significant portion of the cells remain undamaged during the wash and dry procedure. Results from preliminary SIMS measurements show that is it possible to detect a large variety of biomolecular signals, including intact lipids from the plasma membrane in the mass range of 700–900Da from single cells, with little external water interference at the surface.
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| 39. |
- Sharma, Varun, et al.
(författare)
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Revealing Structure and Localization of Steroid Regioisomers through Predictive Fragmentation Patterns in Mass Spectrometry Imaging
- 2023
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 95:48, s. 17843-17850
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Tidskriftsartikel (refereegranskat)abstract
- Identifying and mapping steroids in tissues can provide opportunities for biomarker discovery, the interrogation of disease progression, and new therapeutics. Although separation coupled to mass spectrometry (MS) has emerged as a powerful tool for studying steroids, imaging and annotating steroid isomers remains challenging. Herein, we present a new method based on the fragmentation of silver-cationized steroids in tandem MS, which produces distinctive and consistent fragmentation patterns conferring confidence in steroid annotation at the regioisomeric level without using prior derivatization, separation, or instrumental modification. In addition to predicting the structure of the steroid with isomeric specificity, the method is simple, flexible, and inexpensive, suggesting that the wider community will easily adapt to it. We demonstrate the utility of our approach by visualizing steroids and steroid isomer distributions in mouse brain tissue using silver-doped pneumatically assisted nanospray desorption electrospray ionization mass spectrometry imaging.
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| 40. |
- Thomas, Mathew, et al.
(författare)
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Towards Adaptive, Streaming Analysis of X-ray Tomography Data
- 2015
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Ingår i: Synchrotron Radiation News. - : Informa UK Limited. - 0894-0886 .- 1931-7344. ; 28:2, s. 10-14
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Tidskriftsartikel (refereegranskat)abstract
- Temporal and spatial resolution of chemical imaging methodologies such as X-ray tomography are rapidly increasing, leading to more complex experimental procedures and fast-growing data volumes. Automated analysis pipelines and big data analytics are becoming essential to effectively evaluate the results of such experiments. Offering those data techniques in an adaptive, streaming environment can further substantially improve the scientific discovery process by enabling experimental control and steering based on the evaluation of emerging phenomena as they are observed by the experiment. Pacific Northwest National Laboratory (PNNL)'s Chemical Imaging Initiative (CII, http://imaging.pnnl.gov/) has worked since 2011 towards developing a framework that allows users to rapidly compose and customize high-throughput experimental analysis pipelines for multiple instrument types. The framework, named “Rapid Experimental Analysis” (REXAN) Framework [1M. Thomas, 3D imaging of microbial biofilms: Integration of synchrotron imaging and an interactive visualization interface, Engineering in Medicine and Biology Society (EMBC), 2014 36th Annual International Conference of the IEEE, Chicago, IL, August 28 (2014). [Google Scholar]], is based on the idea of reusable component libraries and utilizes the PNNL-developed collaborative data management and analysis environment “Velo” to provide a user-friendly analysis and data management environment for experimental facilities. This article will discuss the capabilities established for X-ray tomography, review lessons learned, and provide an overview of our more recent work in the Analysis in Motion Initiative (AIM, http://aim.pnnl.gov/) at PNNL to provide REXAN capabilities in a streaming environment.
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| 41. |
- Tóth, Gábor, et al.
(författare)
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Interleukin-13 Treatment of Living Lung Tissue Model Alters the Metabolome and Proteome : A Nano-DESI MS Metabolomics and Shotgun Proteomics Study
- 2024
-
Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 25:9
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Tidskriftsartikel (refereegranskat)abstract
- Asthma is a chronic respiratory disease with one of the largest numbers of cases in the world; thus, constant investigation and technical development are needed to unravel the underlying biochemical mechanisms. In this study, we aimed to develop a nano-DESI MS method for the in vivo characterization of the cellular metabolome. Using air-liquid interface (ALI) cell layers, we studied the role of Interleukin-13 (IL-13) on differentiated lung epithelial cells acting as a lung tissue model. We demonstrate the feasibility of nano-DESI MS for the in vivo monitoring of basal-apical molecular transport, and the subsequent endogenous metabolic response, for the first time. Conserving the integrity of the ALI lung-cell layer enabled us to perform temporally resolved metabolomic characterization followed by "bottom-up" proteomics on the same population of cells. Metabolic remodeling was observed upon histamine and corticosteroid treatment of the IL-13-exposed lung cell monolayers, in correlation with alterations in the proteomic profile. This proof of principle study demonstrates the utility of in vivo nano-DESI MS for characterizing ALI tissue layers, and the new markers identified in our study provide a good starting point for future, larger-scale studies.
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