For single cell proteome analysis, ultra-high sensitivity mass spectrometry is key to reach proteome coverages necessary for understanding the cellular heterogeneity on a cell-by-cell level. Latest enhancements in ion transfer with a larger transfer capillary, an additional higher-pressure segment for more effective ion collection, two orthogonal deflections, to maintain robustness, and high-capacity TIMS pushes the limits of detection to single cell level.
K562 cell digest (Promega) dilution series from 16 ng to 15 pg, were prepared. One, five, ten and twenty HeLa cells and FACS pre-sorted T-Cells (CD4+, CD8+) B-Cells (CD19+) and monocytes (CD14+) from peripheral blood mononuclear cells (PBMCs) were isolated and prepared with a cellenONE (Cellenion). Peptides were loaded onto an Aurora Elite column (IonOpticks) separated with a 22 min active gradient (32SPD) or an Aurora Rapid column (IonOpticks) with a 10 min active gradient (80SPD) using a nanoElute2, eluting peptide detection on a timsTOF Ultra in dia-PASEF, and analysis with Spectronaut 18.
Processing of K562 dilution series acquired in dia-PASEF mode identified >1,000 protein groups out of 15 pg, with 80SPD, and 32SPD and > 6,000 protein groups (80SPD) and >7,000 protein groups (32SPD) for 16 ng. Quantitative accuracy at 250 pg was around 8 - 9% and about 4 - 6% at loads of 4, 8 and 16 ng. Protein groups identified for peptide loads < 500 pg were comparable between 80SPD and 32SPD. Analysis of HeLa cells resulted in about 4,000 protein groups from single cells and up to 6,000 protein groups for 20 cells in 80SPD and 32SPD. For FACS pre-sorted PBMCs, the 80SPD workflow identified in total 1,713 protein groups, with distinct proteomic phenotypes for the four cell types.
Deep proteome coverage and high reproducibility using the timsTOF Ultra combined with automated single cell isolation and sample preparation on the cellenONE® platform.