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72nd ASMS Annual Conference 2024
Mass Analytica posters at 72nd ASMS Annual Conference 2024
Pyxis Unveiled: Advancing Single-Cell MALDI MSI Analysis for Deeper Molecular Insights
Pyxis Unveiled: Advancing Single-Cell MALDI MSI Analysis for Deeper Molecular Insights
72nd ASMS Conference on Mass Spectrometry. June 2024
Ismael Zamora1; Rachelle Balez2; Jayden C. McKinnon2; Reuben S.E. Young2; Liam Robinson2; Lezanne Ooi2; Giuseppe Arturi3; Giulia Sorbi3; Shane Ellis2; Sara Tortorella3
1Mass Analytica, Sant Cugat del Vallès, Spain; 2Molecular Horizons, University of Wollongong, Wollongong, Australia; 3Mass Analytica, Bettona, Italy
Abstract
Introduction
Single-cell metabolomics and lipidomics using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) is emerging as an exciting tool to study metabolic alterations across heterogeneous cell populations. Single-cell analysis using MALDI MSI does however come with technical challenges, including sample preparation workflows compatible with fluorescent microscopy and downstream MS analysis, as well as dedicated data processing workflow to coregister microscopy with MSI and extract profiles from defined cells for later analysis. Here, we introduce an efficient single-cell MSI data analysis pipeline utilising the cutting-edge Pyxis software platform. The performance and versatility of the pipeline are demonstrated by single-cell lipidomics and metabolomics analysis of human-derived astrocytes, unravelling intricate molecular insights at the single-cell level.
Methods
Human astrocytes were generated using induced pluripotent stem cell-derived neurons. Live astrocyte cultures were stained with CellBrite Green and Hoechst, fixed with PFA and washed with cold ammonium acetate prior to microscopy. Following microscopy cells were coated in 2,5-DHA matrix via sublimation for lipid imaging or NEDC matrix using a HTC TM-Sprayer for metabolite imaging for MALDI and MALDI-2 analysis, respectively, using an Orbitrap Elite coupled to a Spectroglyph MALDI/ESI ion source (Spectroglyph LLC, Kennewick, WA, USA). Coregistration with microscopy, segmentation at single-cell level, extraction of spectra generated from single cells, and dedicated statistics to process single-cell spectra were performed using Pyxis (Mass Analytica, Spain). Features of interest were annotated using HMDB and LIPID MAPS databases integrated in Pyxis.
Preliminary data
Rich lipid and metabolite data were recorded from single cells using both MALDI and MALDI-2 with pixel sizes as low as 10 microns. Given the relatively large size of the astrocytes multiple pixels were recorded across single cells, shedding light into sub-cellular metabolite distributions. For example the [M-H]- of adenine was detected predominantly in the soma of the cells.
Ad hoc algorithms were developed and integrated to cope with the data analysis challenges. Rigid and non-rigid strategies to coregister microscopy, fluorescence and MS images were evaluated. An hybrid approach was employed, where rigid registration was applied initially to achieve a coarse alignment, followed by non-rigid registration to refine the alignment at a finer spatial scale. Utilising both approaches in a complementary fashion enhanced the accuracy of spatial integration of multi-modal imaging data. Bisecting k-means and spatial denoising facilitated single-cell border identification. An algorithm was designed to automatically isolate cells, apply user-defined exclusion criteria, calculate single-cell profiles, which then underwent multivariate statistical analysis.
Demonstrated applications of this analytical and data analysis workflow include: (i) single-cell analysis of human-derived astrocyte populations and (ii) investigating the single-cell metabolic and lipidomic responses to inflammatory cytokine stimulation and how these are correlated with cytockeletal remodelling upon inflammatory activation.
By implementing all the necessary data analysis steps into a single, user-friendly software platform, we enable the comprehensive exploration of single-cell MSI data. This integrated approach not only streamlines the intricate process of biochemical data interpretation but also ensures that users can fully exploit the richness of information within their single-cell datasets with ease.
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Revolutionizing Spatial Dermatology: Investigating Sunfilter Efficacy on Reconstructed Human Epidermis with AP-MALDI MSI Metabolomics and Dedicated Data Analysis Software
Revolutionizing Spatial Dermatology: Investigating Sunfilter Efficacy on Reconstructed Human Epidermis with AP-MALDI MSI Metabolomics and Dedicated Data Analysis Software
72nd ASMS Conference on Mass Spectrometry. June 2024
Sara Tortorella1; Maureen Feucherolles2; Giulia Sorbi1; Giuseppe Arturi1; Sue Kennerley3; Gilles Frache2; Ismael Zamora4
1Mass Analytica, Bettona, Italy; 2Luxembourg Institute of Science and Technology, Molecular and Thermal Analysis, Belvaux, Luxembourg; 3K R Analytical, Sandbach, United Kingdom; 4Mass Analytica, Sant Cugat del Vallès, Spain
Abstract
Introduction
Atmospheric Pressure Matrix-Assisted Laser Desorption/Ionization mass spectrometry imaging (AP-MALDI MSI) is a variant of the MALDI technique. The capacity of AP-MALDI MSI to work in an atmospheric environment eliminates the need for vacuum chambers, allowing for the preservation of native hydrated samples as well as the analysis of vacuum-incompatible compounds. This facilitates integration with other analytical techniques and increases sample preparation versatility. It has found applications in many fields such as biology, including spatial dermatology, where the analysis of complex biomolecules is essential. Here we introduce Pyxis, novel vendor neutral software for comprehensive AP-MALDI MSI data analysis, to investigate the spatial lipidome distribution and alteration within sunfilter-protected and -unprotected reconstructed human epidermis (RHE) sections, submitted to UV radiations.
Methods
RHE sections subjected to three test conditions: no UV stress and no sun filter (n=12), UV stress and no sun filter (n=12), and UV stress and sun filter (n=12), were washed, coated with HCCA matrix using the SunCollect MALDI Sprayer (SunChrom GmbH, Germany), and analysed by AP-MALDI MS in both positive and negative ion mode. Here, the compact AP-MALDI (ng) UHR system (MassTech Inc., Columbia, MD), was coupled to a high resolution Orbitrap Exploris 480 mass spectrometer (ThermoFisher, San Jose, CA). Imaging experiments were performed at spatial resolution of 5 µm per pixel, over a mass range of 205–2000 Da and at a mass resolution of 240,000@m/z 200. All data analysis and identification was performed using Pyxis (Mass Analytica, Spain).
Preliminary data
Applications of this unique analytical and data analysis workflow to RHE sections enabled lipids species accumulation within sections to be visualised and identified, providing insight into the metabolomics hallmarks of different sunfilters.
Leveraging the segmentation, based on clustering algorithms, and co-localization capabilities of the Pyxis software, we delineated regions of interest (ROIs) on the RHE sections to perform supervised statistical analyses, i.e. partial least squares discriminant analysis (PLS-DA). The comparison between RHE sections exposed and unexposed to UV light revealed distinctive changes in m/z values across specific cell layers. It allowed us to pinpoint inflammation biomarkers associated with UV exposure. Using these biomarkers, we directly visualised the efficacy of a test sunfilter, annotating the biomarkers, directly within Pyxis, with the integrated LIPID MAPS and HMDB databases. Annotations were ranked using a scoring system, enabling the precise identification of signal and spatial alterations in particular lipid classes, notably sterols. For instance, the oxysterol , 25-hydroxy-cholesterol 3-sulfate, recognized for its involvement in anti-inflammatory response, emerged as a key indicator. While this oxysterol is significantly expressed in UV-exposed and unprotected RHE sections, the opposite was true in the RHE sections not exposed to UV or protected by the sunscreen. Hence, the effectiveness of the sunfilter in mitigating this pathway was discerned by the observed modulation of this lipid. Our findings highlight the utility of the Pyxis platform, providing a user-friendly interface and a full exploitation of comprehensive AP-MALDI MSI data processing workflow, offering insights into metabolomic signatures relevant to dermatological research.
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