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

Background Technological 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. Methods Mouse 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. Results Workflow 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. Conclusions This 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.