Fig. 4: Heterogeneity of single A549 and H460 epithelial-like human lung cancer cells.
a–f, Individual cells of 20–30 µm in diameter were prepared using the One-Pot workflow and analyzed at 50 SPD using the previously optimized settings for LC and MS on the Orbitrap Astral mass spectrometer. First, A549 and H460 cells were analyzed. In a, circles indicate identified PGs or precursors at a 1% FDR of each individual cell, bars indicate mean values, and error bars indicate standard deviations. The search was performed library free with matching across biological replicates but not across all samples; n = 20 individual cells for each cell type. The PCA (b) was based on protein quantities, with each dot representing a cell or blank sample, and heat map clustering (c) was performed with each column representing a cell or blank sample and color codes depicting relative protein abundance. In total, n = 66 A549 cells in predefined size groups of 15–20 µm, 20–25 µm or 25–30 µm in diameter were analyzed (d–f). In d, circles indicate identified PGs at a 1% FDR of each individual cell, bars indicate the mean values, and error bars indicate standard deviations. The search was performed library free in method evaluation mode, DirectDIA+ or DirectDIA+ optimized settings or against a tailored library created from three replicates of 20 or 40 cells. The PCA (e) and UMAP (f) are based on protein quantities (DirectDIA+ analysis) of the 66 A549 cells, with each dot representing a cell and colors reflecting the actual cell size as determined by the camera system of the cellenONE. All samples (a–f) were prepared and measured in the exact same way. Blanks were processed in the same 384-well plate and contained all reagents and buffers but no cells.
