Introduction:
Bottom-up proteomics is rapidly evolving with new instruments and methods demanding continuous evaluation. This study benchmarks the TimsTOF HT and the Orbitrap Eclipse mass spectrometer using three-proteome mixtures (yeast, E. coli, human) in known ratios analyzed with both data-dependent acquisition (DDA) and data-independent acquisition (DIA) workflows.

Methods:
Mixtures of the three proteomes (total protein loads: 0-600 ng) were separated on ionopticks Aurora Ultimate columns and analyzed on TimsTOF HT and Obitrap Eclipse in DDA and DIA modes. DDA data was processed with FragPipe, while DIA data utilized Biognosys™ Spectronaut™. Custom Python scripts (MsReport) were employed for computational analysis.
Results:
Analysis of three replicates per sample mixture in DIA mode on TimsTOF HT identified over 12,000 protein groups (7,800+ human, 2,000+ yeast, 700+ E. coli) and 130,000+ unique peptides, while over 11,000 protein groups were identified with DDA on TimsTOF HT. Notably, >80% of quantified proteins had a coefficient of variation (CV) below 10%, with a median CV of less than 5%. Experimental protein ratios are closely centered around the theoretical ratios. Very few E. coli proteins were quantified in the 0 ng E. coli sample, indicating a low false discovery rate (around 0.4%). DIA identified up to 19% more protein groups than DDA, comprising mostly low-abundant proteins.
Conclusions:
The established workflows offer deep proteome coverage with high accuracy and low false discovery rates, demonstrating their readiness for application to complex biological samples.