Biology subjects

Jakobsen, L. A.

Publications and source records attributed to Jakobsen, L. A..

6 recordsLinked to original sources

bioRxiv2026

STEP-PTMs: Sequential TMT-based Enrichment and Profiling of Post-Translational Modifications

Comprehensive characterization of protein abundance and multiple post-translational modifications (PTMs) from the same biological samples is essential for understanding cellular regulation and PTM crosstalk but remains analytically challenging. Here, we present STEP-PTM (Sequential Tag-based Enrichment of Post-Translational Modifications), a modular TMT-multiplexed workflow that enables integrated quantitative analysis of the proteome, metabolome and multiple PTM classes from a single peptide preparation. Proteins are digested, isobarically labeled using tandem mass tags (TMT), and combined into a single multiplexed peptide pool prior to sequential PTM enrichment, thereby minimizing technical variability, reducing sample requirements and facilitating direct quantitative integration across datasets. STEP-PTM supports flexible sequential enrichment of phosphopeptides, peptides containing free and reversibly modified cysteines, sialylated N-linked glycopeptides, lysine-acetylated peptides and S-palmitoylated peptides, while preserving non-modified peptides for global proteome analysis. PTM-specific database searches further improve identification confidence and quantitative accuracy, and the modular workflow can readily be adapted by incorporating or omitting enrichment modules according to the biological question. Application of STEP-PTM to TMT16-plex cerebral brain organoids enabled the quantification of 10,413 proteins, 2,969 metabolites, 19,655 phosphopeptides, 28,876 peptides containing reversibly modified cysteines, 9,723 peptides containing free cysteines, 1,716 intact sialylated N-linked glycopeptides and 771 lysine-acetylated peptides from the same biological samples. We further demonstrate the applicability of the workflow to multiple mouse tissues, highlighting its broad utility for integrated systems-level characterization of protein expression and PTM regulation across diverse biological models.

biochemistry↗

bioRxiv2026

Patient-Derived Air-Liquid Interface Forebrain Organoids Reveal Functional Synaptic Deficits in Schizophrenia

Schizophrenia (SCZ) is a severe and debilitating neurodevelopmental disorder with lifelong impact on everyday life. Disruptions in synapse functions play a key role in its complex and poorly understood etiological and pathological mechanisms. Here, we investigated both the molecular composition and the spontaneous and stimulated functional properties of synapses in neural organoids from SCZ individuals. Air-liquid interface forebrain organoids (ALI-FOs) were generated from induced pluripotent stem cells (iPSCs) derived from three individuals with SCZ and three healthy controls. At day 170 synaptosomes were enriched and analyzed by data-independent acquisition mass spectrometry to profile the proteome, alongside with TMT-labeled phosphoproteomics both before and after acute KCl-induced depolarization. In parallel, we characterized the PTMome of the surrounding cellular environment, comprising phosphorylation, peptides with free and reversibly modified cysteines, and sialylated N-linked glycopeptides. Functional glutamatergic and GABAergic activity was assessed using calcium imaging to capture spontaneous neuronal signaling. Both conditions exhibited mature synaptic structures, while growth cones were observed only in SCZ-derived ALI-FOs, indicative of ongoing or delayed synaptogenesis. Proteomic analysis of synaptosome preparations revealed 358 differentially regulated proteins between SCZ and controls and 125 phophoproteins with altered phosphorylation, which clustered into three major categories: (1) synaptogenesis and synapse signaling; (2) cytoskeleton and cell junctions; (3) growth cone dynamics and neurite outgrowth. Analysis of the PTMs in the surrounding cellular environment revealed regulation of key regulatory mechanisms in 526 proteins, supporting the synaptic alterations observed. Notably, components of the Wnt signaling pathway were consistently dysregulated across both the synaptosome preparation and the PTMome in SCZ-derived ALIFOs as compared to controls. Depolarization-induced phospho-signaling revealed SCZ-specific response enriched in synaptic vesicle trafficking pathways. Together, these findings provide new insights into early synaptic alterations in SCZ, highlighting changes not only in protein composition, but more in protein regulatory mechanisms underlying synaptic signaling.

neuroscience↗

bioRxiv2025

An ancestral transmembrane transcription factor couples cell envelope regulation and the SOS response in Caulobacter crescentus

The DNA damage (SOS) response in bacteria involves derepression of a set of genes in order to activate mechanisms to tolerate stress, repair DNA and slow down cell division. While some of these genes are well characterized, many genes exist which are clearly induced by DNA damage but for which the function is unclear. In Caulobacter crescentus, the toxin-antitoxin (TA) system higBA and a closely associated downstream transcription factor (higX) are strongly induced as part of the SOS response, but the role of higX is unknown. We show that, unexpectedly, HigX functions independently of HigBA as a cell membrane-associated regulator and is toxic when overexpressed in filamentous cells. ChIP-Seq indicated that it binds to several promoters associated with cell envelope regulation. In cells with the SOS response constitutively activated, HigX was overproduced, but at the same time was unable to bind the majority of its target promoters. higX was highly conserved in genomic context among many alpha-proteobacteria, while higBA was only found upstream of it in a small number of Caulobacter genomes, including the universal laboratory strain C. crescentus NA1000. Compositional analysis suggested that higBA originated from a foreign source, while higX is likely ancestral to the alpha-proteobacteria. Our data support a model where dysregulation of HigX production and activity in filamentous {Delta}lexA cells contributes to cell envelope instability and antibiotic sensitivity. Thus, the protective effect of inhibiting cell division during the SOS response in order to repair the DNA, carries the hidden cost of interference with HigX-mediated cell envelope maintenance. Author summaryThe DNA damage (SOS) response in bacteria is key for their survival in harsh conditions, but the genes which are induced by this response and the molecular mechanisms involved are variable between different bacteria. We investigated the function of an uncharacterized SOS-induced gene in Caulobacter crescentus (higX) and found that it is a membrane-localized DNA binding protein, but that it did not help survival in DNA damage. Instead, it negatively affected cell viability when induced, unlike other SOS response genes. Genomic analysis showed that higX is highly conserved among alphaproteobacteria, but it is only under SOS control in a small number of genomes (including Caulobacter crescentus) where a toxin-antitoxin system and its SOS-regulatory promoter have been inserted upstream. This work uncovers HigX as a novel factor in cell envelope regulation, and shows that loss of cell shape regulation, eg. in filamentous {Delta}lexA cells, can influence the ability of transmembrane transcription factors to bind to their target genes.

microbiology↗

bioRxiv2025

Isolation and characterization of synaptic structures from human neural organoids

Human neural organoids (NOs) provide a powerful platform for investigating synaptic development and dysfunction during early neurodevelopment. However, methodologies for isolating functional synaptic structures from these models remain limited. Here, we present a differential centrifugation protocol enabling the enrichment of growth cone particles (GCPs) and immature synaptosomes from air-liquid interface cerebral organoids (ALI-COs) at distinct developmental stages (day 90 and 150). Notably, the method avoids density gradients, requires minimal starting material while maintaining reproducibility across human and murine tissues. Quantitative proteomic profiling revealed significant enrichment of growth cone markers (e.g. GAP43) and classical synaptosomal proteins (e.g. PCLO, BSN, SYN1). Transmission electron microscopy (TEM) confirmed the presence of membrane-enclosed GCPs with fibrous content and mitochondria in day 90 isolates, and immature synaptosomes containing synaptic vesicles on day 150. Functional viability of both types of synaptic structures was demonstrated through KCl-induced depolarization, which triggered phosphorylation changes in growth cone proteins (GAP43, MARCKS, MARCKSL1), cytoskeletal regulators (DCLK1, SHTN1, MARK4, MAP1B) and protein kinases (CAMK2G, PRKCE) in day 90 GCPs, as well as classical synaptic vesicle cycle proteins (SYN1, DNM1, RPH3A) at day 150. Overall, this study establishes a centrifugation-based protocol for isolating growth cones and immature synapses from human organoids, capturing key stages of synaptic development and enabling scalable, patient-compatible models to study synaptic function and dysfunction in neurodevelopmental and neurodegenerative disorders.

neuroscience↗

bioRxiv2024

Temporal proteomic and PTMomic atlas of cerebral organoid development

Cerebral organoids (CBOs) are generated from pluripotent stem cells that undergo neuroectoderm specification and neuronal differentiation in three dimensions. The developing neurons in CBOs migrate and self-organize into cerebral cortex-like layers, mimicking human brain development. CBOs develop according to intrinsic signaling mechanisms and offer unique insights into mechanisms of early human brain development. This process requires coordinated spatiotemporal regulation of protein expression and function, where the latter can be achieved by post-translational modifications (PTMs) on proteins. Despite the importance of proteins in brain development and function, profiling of protein abundance and the involvement of PTMs in CBO development remain underexplored. To gain insight into protein and PTM abundance in CBOs, we performed a high-resolution temporal analysis of CBOs up to day 200 using proteomics, PTMomics and metabolomics. We quantified more than 9,300 proteins and various neurodevelopmentally relevant PTMs (including phosphorylation, lysine acetylation, sialylated N-glycosylation, and cysteine modifications). We demonstrate that protein abundance and dynamic PTMs show significant temporal changes during CBO development related to neuronal differentiation and energy metabolism, whereas calcium signaling is mainly regulated by dynamic PTMs. We further show that synaptic protein content correlated with neurotransmitter levels, and we detected astroglia beyond day 100. Lastly, comparative analysis showed proteomic similarities between CBOs and human fetal brain tissue, supporting the physiological relevance of CBOs. Overall, our study presents a temporal atlas of protein and PTM abundance in CBOs and provides a valuable resource for studying neurodevelopment in neural organoids.

neuroscience↗

bioRxiv2023

Multi-omic analysis of guided and unguided forebrain organoids reveal differences in cellular composition and metabolic profiles

Neural organoids are invaluable model systems for studying neurodevelopment and neurological diseases. For this purpose, reproducible differentiation protocols are needed that minimize inter-organoid variability whilst generating neural organoids that physiologically resemble the brain area of interest. Currently, two main approaches are used: guided, where the differentiation towards neuroectoderm and subsequently specific CNS regions is driven by applying extrinsic signalling molecules, and unguided, where the intrinsic capability of pluripotent stem cells to generate neuroectoderm without external signalling is promoted. Despite the importance for the field, the resulting differences between these models have not been directly investigated. To obtain an unbiased comparison, we performed a multi-omic analysis of forebrain organoids generated using a guided and unguided approach focusing on proteomic, lipidomic and metabolomic differences. Furthermore, we characterised differences in phosphorylation and sialylation states of proteins, two key post-translational modifications (PTMs) in neurodevelopment, and performed single cell transcriptomics (scRNAseq). The multi-omic analysis revealed considerable differences in neuronal-, synaptic and glial content, indicating that guided forebrain organoids contain a larger proportion of neurons, including GABAergic interneurons, and synapses whereas unguided organoids contain significantly more GFAP+ cells and choroid plexus. Furthermore, substantial differences in mitochondrial- and metabolic profiles were identified, pointing to increased levels of oxidative phosphorylation and fatty acid {beta}-oxidation in unguided forebrain organoids and a higher reliance on glycolysis in guided forebrain organoids. Overall, our study comprises a thorough description of the multi-omic differences arising when generating guided and unguided forebrain organoids and provide an important resource for the organoid field studying neurodevelopment and -disease.

neuroscience↗