Biology subjects

Enzlein, T.

Publications and source records attributed to Enzlein, T..

7 recordsLinked to original sources

bioRxiv2025

A sulfatide-centered ultra-high resolution magnetic resonance MALDI imaging benchmark dataset for MS1-based lipid annotation tools

Spatial omics techniques are indispensable for studying complex biological systems and for the discovery of spatial biomarkers. While several current matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) instruments are capable of localizing numerous metabolites at high spatial and spectral resolution, the majority of MSI data is acquired at the MS1 level only. Assigning molecular identities based on MS1 data presents significant analytical and computational challenges, as the inherent limitations of MS1 data preclude confident annotations beyond the sum formula level. To enable future advancements of computational lipid annotation tools, well-characterized benchmark - or ground truth - datasets are crucial, which exceed the scope of synthetic data or data derived from mimetic tissue models. To this end, we provide two sulfatide-centered, biology-driven magnetic resonance MSI (MR-MSI) datasets at different mass resolving powers that characterize lipids in a mouse model of human metachromatic dystrophy. This data includes an ultra-high-resolution (R [~]1,230,000) quantum cascade laser mid-infrared imaging-guided MR-MSI dataset that enables isotopic fine structure analysis and therefore enhances the level of confidence substantially. To highlight the usefulness of the data, we compared 118 manual sulfatide annotations with the number of decoy database-controlled sulfatide annotations performed in Metaspace (67 at FDR < 10%). Overall, our datasets can be used to benchmark annotation algorithms, validate spatial biomarker discovery pipelines, and serve as a reference for future studies that explore sulfatide metabolism and its spatial regulation.

biochemistry↗

bioRxiv2024

Exploring the Abeta Plaque Microenvironment in Alzheimer's Disease Mice by Multimodal Lipid-Protein-Histology Imaging on a Benchtop Mass Spectrometer

Amyloid-{beta} (A{beta}) plaque deposits in the brain are a hallmark of Alzheimers disease (AD) neuropathology. Plaques consist of complex mixtures of peptides like A{beta}1-42, characteristic lipids such as gangliosides, and they are targeted by reactive microglia and astrocytes. In pharmaceutical research and development it is therefore a formidable challenge to contextualize different biomolecular classes and cell types of the A{beta} plaque microenvironment in a coherent experimental workflow on a single tissue section and on a benchtop imaging reader. Here, we developed such a workflow that combines lipid MALDI mass spectrometry imaging using a vacuum-stable matrix with histopathology stains and with MALDI HiPLEX-immunohistochemistry of plaques and multiple protein markers on a bench-top imaging mass spectrometer. The three data layers consisting of lipid, protein markers, and histology can be co-registered and evaluated together. Multimodal data analysis suggests extensive co-localization of A{beta} plaques with the peptide precursor protein, with a defined subset of lipids and with reactive glia cells on a single brain section of APP/PS1 AD model mice. Plaque-associated lipids like ganglioside GM2 and phosphatidylinositol PI38:4 isoforms were readily identified using the tandem MS-capabilities of the mass spectrometer. Taken together, our data suggests that complex pathology involving multiple lipids, proteins and cell types can be interrogated by this spatial multiomics workflow on an easy-to-use benchtop mass spectrometer.

neuroscience↗

bioRxiv2024

Label-free Assessment of Complement-Dependent Cytotoxicity of Therapeutic Antibodies via a Whole-Cell MALDI Mass Spectrometry Bioassay

Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and bench-marked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration-response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.

biochemistry↗

bioRxiv2023

Deep MALDI-MS Spatial Omics guided by Quantum Cascade Laser Mid-infrared Imaging Microscopy

In spatial omics, highly confident molecular identifications are indispensable for the investigation of complex biology and for spatial biomarker discovery. However, current mass spectrometry (MS)-based spatial omics must compromise between data acquisition speed and biochemical profiling depth, thus often leading to only "putative" molecular identifications. Here, we introduce fast quantum cascade laser mid-infrared imaging microscopy to guide MS imaging to confined tissue areas of high interest, e.g., multicellular spheroid cores or kidney glomeruli, for spatial lipidomics profiling at maximized analytical depth utilizing magnetic resonance-MS imaging at >106 resolution or prm-PASEF-MS2 fragmentation imaging. Instigating selective sulfatide accumulation in arylsulfatase A-deficient mice as ground truth concept, we demonstrate that deep QCL-infrared-guided on-tissue spatial omics unequivocally identifies 120 sulfatides. This approach enables identifications of odd-chain sulfatides and studies of structure-ion mobility-relationships that provide chemical rationales for improvements to current ion mobility prediction algorithms. Workflows and data processing tools are provided as community resources.

biochemistry↗

bioRxiv2023

APP substrate ectodomain defines A beta length by restraining gamma-secretase processivity and facilitating product release

Sequential proteolysis of the amyloid precursor protein (APP) by {gamma}-secretases (GSECs) generates amyloid-{beta} (A{beta}) and defines the proportion of short-to-long A{beta} peptides, which is tightly connected to Alzheimers disease (AD) pathogenesis. Here, we study the mechanism controlling substrate processing by GSECs and defining product length. We found that polar interactions established by the APPC99 ectodomain (ECD), involving but not limited to its juxtamembrane region, restrain both the extent and degree of GSEC processive cleavage by destabilizing enzyme-substrate (E-S) interactions. We show that increasing hydrophobicity at APPC99-ECD - due to mutation or ligand binding - attenuates this substrate-driven product release mechanism, and rescues the effects that AD pathogenic variants exert on A{beta} profiles. In addition, our study reveals that APPC99-ECD facilitates the paradoxical production of longer A{beta}s caused by some GSEC inhibitors that act as high-affinity competitors to the substrate. These findings assign a pivotal role to the substrate ECD in the sequential proteolysis by GSEC and suggest it as a sweet spot for the potential design of APP targeting compounds selectively promoting its processing by GSEC.

biochemistry↗

bioRxiv2023

Alzheimer's disease linked Aβ42 exerts product feedback inhibition on γsecretase impairing downstream cell signaling

Amyloid {beta} (A{beta}) peptides accumulating in the brain are proposed to trigger Alzheimers disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for A{beta}42 toxicity that arises from its proven affinity for {gamma}-secretases. We hypothesized that the reported increases in A{beta}42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on {gamma}-secretases, and thereby impair downstream signaling events. We show that human A{beta}42 peptides, but neither murine A{beta}42 nor human A{beta}17-42 (p3), inhibit {gamma}-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75 and pan-cadherin. Moreover, A{beta}42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in A{beta}42 contribute to cellular toxicity via the {gamma}-secretase inhibition, and provide a novel conceptual framework to address A{beta} toxicity in the context of {gamma}-secretase-dependent homeostatic signaling.

biochemistry↗

bioRxiv2020

Imaging stable isotope labelling kinetics (iSILK) for following spatial Aβ plaque aggregation dynamics in evolving Alzheimers disease pathology

For our understanding of Alzheimers disease (AD) pathophysiology, it is of critical importance to determine how key pathological factors, specifically amyloid {beta} (A{beta}) plaque formation, are interconnected and implicated in neurodegeneration, disease progression and the development of clinical symptoms. Exactly how A{beta} plaque formation is initiated and how the ongoing plaque deposition proceeds is not well understood. This is partly because we can only examine details of the molecular pathology after death in humans, and in mice, we can only examine a particular point in time without any longitudinal information on the fate of individually formed deposits. Herein, we used metabolic labelling of proteins with stable isotopes, together with multimodal imaging mass spectrometry (IMS) for imaging stable isotope labelling kinetics (iSILK) in the APPNL-G-F knock-in mouse model of AD. The aim was to monitor the earliest seeds of A{beta} deposition through ongoing plaque development and track the deposition of A{beta} that is produced later in relation to already deposited plaques. This allowed us to visualize A{beta} peptide aggregation dynamics within individual deposits across different brain regions. We identified the cortex as a primary site of deposition in precipitating plaque pathology. Further, our data show that structural plaque heterogeneity is associated with differential peptide deposition. Specifically, A{beta}1-42 is forming an initial core seed followed by radial outgrowth and late secretion and deposition of A{beta}1-38. Together these data prove the potential of iSILK for probing amyloid protein secretion, processing and aggregation dynamics in AD pathology.

neuroscience↗