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bioRxiv · 10.1101/2025.11.02.686128

Investigation of Neurons and Microglia from APOE3 Induced Pluripotent Stem Cells using Data-Independent Acquisitions on a ZenoTOF 7600

Burton, J. B.·McHugh, T.·Schurman, C. A.·Bons, J.·Ellerby, L.·Schilling, B.
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Abstract

Induced pluripotent stem cell (iPSC)-derived neurons and microglia are valuable human models for studying neurodegenerative diseases. Specifically, the apolipoprotein E4 (APOE4) gene is a major genetic risk factor for late-onset Alzheimers disease. Apolipoprotein E (APOE) alleles E2, E3 and E4 can be beneficial, neutral, or increase the risk of Alzheimers disease (AD). Here, we developed a proteomic workflow using data-independent acquisitions to provide a quantitative mass spectrometric proteome analysis, and proteomic screening assays for brain-specific cell types derived from iPSC. Protein groups were quantified in APOE3 neurons and microglia, respectively, with [~]80% overlap. Cell type-specific markers and enriched pathways reflected the specialized functions of each cell type, such as synaptic signaling in neurons and immune and inflammatory responses in microglia. The neuron-specific markers included proteins APP, CALB1, CALB2, DLGs, GAP43, NEFL, MAPs; while microglial markers included proteins AIF1, CDs, MMP9, and ITGAM. Ultimately, the combination of robust iPSC differentiation and sensitive proteomic screening assays described here provides a valuable platform for probing the cellular mechanisms underlying neurological disorders. SignificanceThe quantification of dysregulated proteins and pathways in patient-derived neurons and microglia can provide insights into disease etiology and progression. More broadly, this DIA approach enables deep proteome profiling of unique iPSC-derived cell models, increasing their utility for investigating disease biology and therapeutic development. We focused on iPSC models from two important cell types of the brain, excitatory neurons and microglia. We integrated the proteomes of these two cell types. These tools provide robust biological and mass spectrometric screening tools for future therapeutic interventions using disease-relevant human brain cell types or brain organoid models. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/686128v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d7dorg.highwire.dtl.DTLVardef@17594d3org.highwire.dtl.DTLVardef@7dd894org.highwire.dtl.DTLVardef@1606db_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPresentation of a proteomic workflow using data-independent acquisitions to monitor and screen proteomes of iPSC-derived brain cell types. C_LIO_LIQuick MS Assays to determine protein profiles of iPSC-derived neurons and microglia. C_LIO_LICharacterization of different cell type proteomes from APOE3 iPSCs. C_LIO_LIRevealing of neuron-specific markers and microglia-specific markers by mass spectrometry. C_LIO_LIStep-by-step instructions for the set-up of the DIA-MS assays C_LI

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Burton, J. B., McHugh, T., Schurman, C. A., Bons, J., Ellerby, L., Schilling, B.. 2025-11-04. Investigation of Neurons and Microglia from APOE3 Induced Pluripotent Stem Cells using Data-Independent Acquisitions on a ZenoTOF 7600. https://doi.org/10.1101/2025.11.02.686128

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