Increasing throughput while maintaining coverage depths in single cell proteomics using the timsTOF Ultra

Introduction (119 of 120 words)

For single cell proteome analysis, throughput while maintaining coverage depths is one of the bottlenecks in the field. Therefore, fast scanning, ultra-high sensitivity mass spectrometry is a key to reach a proteome coverage necessary for understanding the cellular heterogeneity on a cell-by-cell level across large sample cohorts. The latest enhancements in 4D-proteomicsTM on the timsTOF platform with high ion intake, a higher-pressure segment for more effective ion collection and two orthogonal deflections, to maintain robustness, pushes the limits of detection to the single cell level. Combined with automated single cell isolation and sample preparation on the cellenONE® platform for protein-loss reduced preparation with direct sample pickup from the label-free proteoCHIP® enables deep proteome coverage and high reproducibility.

Methods (120 of 120 words)

HeLa cells at different cell counts were isolated into the LF48 proteoCHIP®, directly lysed and digested using the cellenONE platform. Samples were either dried down in the chip or manually transferred into 96-well plates. Peripheral blood mononuclear cells (PBMCs) were FACS sorted into T-Cells (CD4+, CD8+) B-Cells (CD19+) and monocytes (CD14+), isolated with the cellenONE and prepared as the HeLa cells above. The LF48 proteoCHIP was placed into the nanoElute® 2 autosampler. Sample were picked up with the dissolve sample function and injection onto a 5 cm Aurora Rapid 75 or a 25 cm Aurora Ultimate C18 column (IonOpticks) and eluted into a timsTOF Ultra at 80 or 32 SPD. dia-PASEF® data were processed with Spectronaut 18 (Biognosys) using directDIA+.

Preliminary Data (201 of 300 words)

A K562 protein extract digests (Promega) dilution series were run on both column setups. Processing of the dia-PASEF data with Spectronaut 18 demonstrated good quantitative reproducibility for individual concentration loads. For loads of les than 500 pg the 80 SPD (10 min active gradient), and 32 SPD (22 min active gradient. Showed same protein coverage depths only for the high loads > 2 ng, the 32 SPD setup outperformed the faster 80SPD setup. Therefore, the 80 SPD setup was used for the following experiments.

Sample pickup directly from the label-free proteoCHIP with the dissolve sample function of the nanoElute® 2 was compared to proteome depths obtainable with samples manually transferred to 96 well plates. Both setups performed equally well delivering a protein depth of about 5,000 proteins per single cell and increasing to >7,000 proteins for 20 cells.

The PBMC analysis Workflow from FACS sorted T-Cells (CD4+, CD8+) B-Cells (CD19+) and monocytes (CD14+) in the 80 SPD setup processed with Spectronaut 18 in directDIA+ identifying over 2,000 protein.

groups across the different cell types with good group identification rate and quantitation reproducibility. Clear differences were observed, distinguishing the two T-Cells classes from each other and also from the B-Cells and monocytes.