Scalability while maintaining coverage depths a bottleneck in single cell proteomics. Fast scanning, ultra-high sensitivity mass spectrometry is a key to reach a proteome coverage necessary for understanding cellular heterogeneity across large sample cohorts. Here we use the cellenONE® platform with an oil-free LF48 proteoCHIP for direct sample pickup using the nanoElute 2 and detection with a timsTOF Ultra 2 to investigate peripheral blood mononuclear cells (PBMCs).
PBMCs were FACS sorted and isolated with the cellenONE at single cell level and 10 cell bulks, then directly lysed and digested. The LF48 proteoCHIP was placed into the nanoElute® 2 autosampler. Sample were picked up using the dissolve sample function, injected onto a 5 cm Aurora Rapid 75 C18 column (IonOpticks) and eluted into a timsTOF Ultra 2 at a speed of ~80 SPD. dia-PASEF® data were processed with Spectronaut 19 using directDIA+.
Ten cell bulks per cell type were used to generate a spectral library resulting in about 4400 protein groups that was used for protein extraction. We identified ~3,000 proteins from 10 cells bulks of CD4+ and CD8+ T-cells and monocytes. B-cells demonstrated lower protein IDs of ~2,000 protein groups in the 10-cell bulks. Similar trends were seen for single cells, with more than 2,000 proteins for CD4+ , CD8+ T-cells and monocytes, with ~1,800 proteins for the B-cells. On a quantitative level, single cells showed high reproducibility within cell type groups, but demonstrated clear differences between the different cell classes, distinguishing the two T-cell classes from each other and from B-cells and monocytes. Unsorted PBMCs (48 cells in total) demonstrated a similar proteome depth. However, as the T-cell population was dominating in this experiment, no clear sub-class grouping was obtained.
Label-free analysis of single PBMCs using the timTOF Ultra 2 platform identifies more than 2000 protein groups per cell.