Microarray and Mass Spectrometry-Based Methodology for Lipid Profiling of Tissues and Cell Cultures

Fernández, Roberto; Garate, José Francisco Valencia; Tolentino-Cortez, Tarson; Herraiz, Ainara; Lombardero, Laura; Ducrocq, Fabien; Rodrı́guez-Puertas, Rafael; Trifilieff, Pierre; Astigarraga, Egoitz; Barreda-Gómez, Gabriel; Fernández, José A.

doi:10.1021/acs.analchem.9b04529

Cited by 22 publications

(33 citation statements)

References 52 publications

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“…In this sense, a cell membrane microarray (CMM) has been successfully used in the study of membrane proteins not only at expression level but also for functional activity [20,21]. As the whole membrane is printed onto these CMMs, the integrity of the proteins remains stable for long periods of time, mainly due to the conservation of the lipid environment [22,23]. Moreover, regional distribution of cholinesterase and specific lipids has been described using CMMs [22].…”

Section: Introductionmentioning

confidence: 99%

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Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Rienda¹,

Elexpe

Tolentino-Cortez³

et al. 2021

Analytica

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Acetylcholinesterase (AChE) is responsible for hydrolyzing the acetylcholine neurotransmitter, bringing an end point to cholinergic neurotransmission. Thus, AChE is the primary target of a wide spectrum of compounds used as pesticides, nerve agents or therapeutic drugs for neurodegenerative diseases such as Alzheimer’s disease (AD). This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. Therefore, the aim of this work is to report a novel technology that enables the simultaneous determination of tissue specific AChE activity, as well as the analysis and screening of specific inhibitors, by using cell membrane microarrays. These microarrays were composed of cell membranes, isolated from 41 tissues, organs and brain areas, that were immobilized over a slide, maintaining the functionality of membrane proteins. To validate this platform, demonstrating its usefulness in drug discovery as a high throughput screening tool, a colorimetric protocol to detect the membrane-bound AChE activity was optimized. Thus, rat cortical and striatal AChE activities were estimated in presence of increased concentrations of AChE inhibitors, and the donepezil effect was assessed simultaneously in 41 tissues and organs, demonstrating the major potential of this microarray’s technology.

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Section: Introductionmentioning

confidence: 99%

“…As the whole membrane is printed onto these CMMs, the integrity of the proteins remains stable for long periods of time, mainly due to the conservation of the lipid environment [22,23]. Moreover, regional distribution of cholinesterase and specific lipids has been described using CMMs [22].…”

Section: Introductionmentioning

confidence: 99%

Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Rienda¹,

Elexpe

Tolentino-Cortez³

et al. 2021

Analytica

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“…Likewise, sulfatides are detected in negative-ion mode, and any interference between PC and PE is avoided. This analysis was performed as described previously 59 , whereby a LTQ-Orbitrap XL analyzer (ThermoFisher, San Jose, CA, USA) equipped with an N2 laser (100 µJ max power, elliptical spot, 60 Hz repetition rate) scanned the material in negative ion mode at a spatial resolution between 15 and 25 µm. A mass resolution of 60,000 and 100,000 was used to record the data from the full scan spectra, and that of around 2000 for MS/MS and MS 3 due to the higher sensitivity of the Ion Trap (IT).…”

Section: Methodsmentioning

confidence: 99%

Comparative lipidomic analysis of mammalian retinal ganglion cells and Müller glia in situ and in vitro using High-Resolution Imaging Mass Spectrometry

Pereiro

Fernández

Barreda-Gómez³

et al. 2020

Sci Rep

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In order to better understand retinal physiology, alterations to which underlie some ocular diseases, we set out to establish the lipid signature of two fundamental cell types in the retina, Müller Glia and Retinal Ganglion Cells (RGCs). Moreover, we compared the lipid signature of these cells in sections (in situ), as well as after culturing the cells and isolating their cell membranes (in vitro). The lipidome of Müller glia and RGCs was analyzed in porcine retinal sections using Matrix Assisted Laser Desorption Ionization Imaging Mass Spectrometry (MALDI-IMS). Isolated membranes, as well as whole cells from primary cell cultures of RGCs and Müller glia, were printed onto glass slides using a non-contact microarrayer (Nano Plotter), and a LTQ-Orbitrap XL analyzer was used to scan the samples in negative ion mode, thereafter identifying the RGCs and Müller cells immunohistochemically. The spectra acquired were aligned and normalized against the total ion current, and a statistical analysis was carried out to select the lipids specific to each cell type in the retinal sections and microarrays. The peaks of interest were identified by MS/MS analysis. A cluster analysis of the MS spectra obtained from the retinal sections identified regions containing RGCs and Müller glia, as confirmed by immunohistochemistry in the same sections. The relative density of certain lipids differed significantly (p-value ≤ 0.05) between the areas containing Müller glia and RGCs. Likewise, different densities of lipids were evident between the RGC and Müller glia cultures in vitro. Finally, a comparative analysis of the lipid profiles in the retinal sections and microarrays identified six peaks that corresponded to a collection of 10 lipids characteristic of retinal cells. These lipids were identified by MS/MS. The analyses performed on the RGC layer of the retina, on RGCs in culture and using cell membrane microarrays of RGCs indicate that the lipid composition of the retina detected in sections is preserved in primary cell cultures. Specific lipid species were found in RGCs and Müller glia, allowing both cell types to be identified by a lipid fingerprint. Further studies into these specific lipids and of their behavior in pathological conditions may well help identify novel therapeutic targets for ocular diseases.

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“…Microarray technology allows the immobilization of cell membranes, antibodies, enzymes and other proteins, as well as whole cells, on different surfaces without disrupting their functional activity. Therefore, they are very versatile tools for immunochemistry, autoradiography, radioligand-binding studies, mitochondrial toxicity assays [124][125][126] or other approaches such as colorimetric and mass spectrometry techniques [126,127]. Microarrays allow a reduction of the number of samples, drugs, chemicals and radioactive residues.…”

Section: Monitorization Of Patients' Immunoresponsesmentioning

confidence: 99%

Enzyme Therapy: Current Challenges and Future Perspectives

Fuente

Lombardero²,

Gómez-González

et al. 2021

IJMS

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In recent years, enzymes have risen as promising therapeutic tools for different pathologies, from metabolic deficiencies, such as fibrosis conditions, ocular pathologies or joint problems, to cancer or cardiovascular diseases. Treatments based on the catalytic activity of enzymes are able to convert a wide range of target molecules to restore the correct physiological metabolism. These treatments present several advantages compared to established therapeutic approaches thanks to their affinity and specificity properties. However, enzymes present some challenges, such as short in vivo half-life, lack of targeted action and, in particular, patient immune system reaction against the enzyme. For this reason, it is important to monitor serum immune response during treatment. This can be achieved by conventional techniques (ELISA) but also by new promising tools such as microarrays. These assays have gained popularity due to their high-throughput analysis capacity, their simplicity, and their potential to monitor the immune response of patients during enzyme therapies. In this growing field, research is still ongoing to solve current health problems such as COVID-19. Currently, promising therapeutic alternatives using the angiotensin-converting enzyme 2 (ACE2) are being studied to treat COVID-19.

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Microarray and Mass Spectrometry-Based Methodology for Lipid Profiling of Tissues and Cell Cultures

Cited by 22 publications

References 52 publications

Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Comparative lipidomic analysis of mammalian retinal ganglion cells and Müller glia in situ and in vitro using High-Resolution Imaging Mass Spectrometry

Enzyme Therapy: Current Challenges and Future Perspectives

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