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bioRxiv · 10.1101/2025.09.23.678003

Single-cell proteomics: a powerful new tool to study kidney cell heterogeneity

Wu, Q.·Cheng, L.·Assmus, A. M.·Zheng, X.·Esteva Font, C.·Petersen, C. C.·Ding, X.·Paludan, S. R.·Fenton, R. A.
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Abstract

BackgroundTechnological advancements in protein mass spectrometry have significantly enhanced analytical sensitivity and throughput, enabling single-cell proteomics by mass spectrometry (SCP-MS) to become reality. SCP-MS allows high-resolution analysis of cellular heterogeneity and function, bypassing bulk analysis limitations. Here, we used SCP-MS to document at the protein level the cellular diversity of mouse kidney cells. We further focused SCP-MS on low abundance distal convoluted tubule (DCT) cells that are essential for body electrolyte homeostasis and blood pressure control. MethodsMouse kidney cells were isolated via enzymic digestion and single-cells isolated using fluorescence- activated cell sorting (FACS). DCT cells were isolated from mice with GFP expression specifically in the DCT (parvalbumin-GFP) using a similar workflow. SCP-MS analyses was performed using an Orbitrap Ascend Mass Spectrometer with a FAIMS Pro Duo interface coupled with Wide ISolation window High-resolution MS1 Data Independent Acquisition (WISH-DIA). Spectronaut was used for protein identification and quantification, and cell-type annotation and clustering were performed in Python by scanpy implementation. ResultsWorkflow benchmarking using Hela cells confirmed a successful SCP-MS setup. From 768 single mouse kidney cells, SCP-MS identified 2626 unique proteins. Computational approaches resolved various nephron segments, including distinct DCT populations. From enriched DCT cells, 1912 proteins were identified, enabling classification of three populations - DCT1, DCT2, and a proliferative subset (ProLIF) that represented a transient state between DCT1 and DCT2. ProLIF cells had elevated abundance of the sodium-chloride cotransporter NCC and represented approximately 33% of DCT cells, substantially exceeding previous transcriptomic estimates of ProLIF cell frequency (~0.1%). High proliferation (~39%) of DCT cells was confirmed using immunohistochemistry. ConclusionsThis SCP-MS analysis of mouse kidney uncovered significant cellular heterogeneity not captured effectively using transcriptomics. Despite imitations in proteome depth and throughput, SCP-MS provides a powerful approach for investigating kidney cellular dynamics at the protein level. Key pointsO_LIWe developed a single-cell proteomics by mass spectrometry (SCP-MS) workflow identifying up to ~2,000 proteins in a single mouse kidney cell C_LIO_LISCP-MS identified a substantial proliferative subset of cells in the DCT underestimated by transcriptomics C_LIO_LISCP-MS could inform on clinical strategies for kidney disease monitoring, offering robust indicators of tubular injury or regeneration. C_LI

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Wu, Q., Cheng, L., Assmus, A. M., Zheng, X., Esteva Font, C., Petersen, C. C., Ding, X., Paludan, S. R., Fenton, R. A.. 2025-09-23. Single-cell proteomics: a powerful new tool to study kidney cell heterogeneity. https://doi.org/10.1101/2025.09.23.678003

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