Advances in ultra-high sensitivity MS and ultra-fast chromatography enabled combination of depth and sample throughput required for large clinical cohort studies. Fast scan speeds and proven robustness of the timsTOF platform enables high analyte sampling for accurate quantification with deep proteome coverage. Here, we assess performance, scalability, and reproducibility of the new standard methods with throughput from 30 to 500SPD on the Evosep Eno system using a timsUltra AIP.
Dilution series of HeLa, single FaDu cells (Head and Neck squamous cell carcinoma, NSCC), and laser micro-dissected human FFPE tonsil tissue of T-cell, B-cell, and squamous cell epithelium were benchmarked. HeLa digest was loaded on Evotips at 250pg up to 100ng. The FFPE tonsil tissue (30,000µm²) were collected into proteoCHIP EVO96 and prepared using a cellenONE. Samples acquisition in dia-PASEF mode on a timsUltra AIP at 500, 300 200, 100, 60 and 30SPD on the Evosep Eno.
The new standard methods on the Evosep Eno significantly improved protein and peptide identification. Evaluation with HeLa peptide dilutions revealed from the lowest HeLa peptide load, 1,500 protein groups at 500SPD and > 4,500 at 30SPD. 100 ng identified > 5,000 at 500SPD protein groups and > 8,500 at 30SPD.
FaDu cells showed increased protein identifications from an average of 3,500 at Whisper Zoom 120SPD to 5,100 at Whisper Zoom 40SPD. Standard gradients yielded between 1,500 (500SPD) and 4,100 (30SPD) protein groups
Tissue contours from Human tonsil resulted in ~2,000 protein groups for the different cell type niches at 500SPD and increased to 6,000 protein groups at 30SPD. Biological differences between cell types were preserved at all gradient lengths with good quantitative performance. Among differentiating proteins, well-known markers were identified (e.g. epithelium: Cadherin1, T-cells: CD3: B-cells: CD19), demonstrating excellent scalability and reproducibility of the Evosep Eno timsUltra AIP workflow, enabling comprehensive large cohort investigations.