Expanding the Limits Single Glomerulus Measurements by Targeted, Quantitative Proteomics

Background: As age progresses, the kidney glomeruli can undergo heterogeneous structural changes that affect filtration efficiency and cause kidney dysfunction. To capture these structural changes at the proteomics level, single glomerulus measurements need to be performed to see the spatial heterogeneity that would otherwise be lost in aggregated, bulk kidney or glomerular samples. We aim to improve the sensitivity and sample recovery of a single glomerulus to better profile its molecular heterogeneity. Methods: Single glomerulus samples were individually dissected from a pool for bulk glomerulus samples from a 7 month old mouse. An optimized acid lysis protocol was performed prior to protein aggregate capture (PAC) and clean-up. A targeted proteomics method was generated from a data-independent acquisition (DIA) library of pooled bulk glomerulus samples. Our targeted list includes peptides from podocyte, glomerular and tubule proteins. Proteomics data on all of the samples were acquired using a targeted method measuring 571 peptides from 500 proteins on an EvoSep running a 30 minute liquid chromatography gradient coupled to a Lumos tribrid mass spectrometer. Results: We applied our optimized targeted protocol to study 30x single mouse glomeruli compared to bulk glomeruli. Podocyte and glomerular proteins, such as Synaptopodin and Podocin, were enriched in protein abundance, and tubule proteins, such as Uromodulin, were highly depleted in the single glomerulus samples compared to the bulk glomerulus control. To assess heterogeneity of the single glomerulus samples, the coefficient of variation was calculated at the peptide level. A higher coefficient of variation (62%) was observed for the single glomerulus samples compared to the bulk glomerulus samples (20%), demonstrating biological variation of individually measured single glomeruli compared to pooled bulk glomerulus samples. Conclusion: We developed an effective sample preparation strategy to recover peptides down to the single glomerulus level. Using massively parallel targeted proteomics, we can measure 571 peptides from both single glomeruli and bulk samples with robust quantitation and no missing data. This method allows for the capture of robust individual glomerular proteomes and captures the confounding heterogeneity that accompanies glomerular aging.