Introduction
Deciphering the molecular intricacies of biological systems demands analytical tools that offer both high sensitivity and broad dynamic range for protein characterization. The newly developed timsOmni instrument features a sophisticated ion manipulation system that supports multiple ion activation and dissociation methods (MSⁿ, eXd), making it particularly well-suited for top-down proteomics. This enables comprehensive structural and functional analysis of intact proteins and proteoforms. Although optimized for advanced applications, timsOmni is built upon the timsUltra AIP platform, originally designed for bottom-up proteomics, and delivers exceptional sensitivity ideal for single-cell and other low-input workflows. This study explores the shotgun proteomics performance of timsOmni, utilizing the omnitrap module in pass-through configuration.
Methods
To assess sensitivity, we performed serial dilutions of tryptic digests from human cell lines (HeLa/K562), reaching down to picogram quantities. Quantitative accuracy was evaluated using mixtures of HeLa, S. cerevisiae, and E. coli digests combined in defined ratios. Sample separation was achieved via nanoHPLC (nanoElute 2) paired with a captivespray ultra ionization source. Data acquisition employed dia-PASEF mode, with subsequent analysis conducted using Spectronaut version 20 (Biognosys) and DIA-NN 2.0 (Aptila).
Results
Initial experiments using 22-minute nanoLC gradients identified roughly 5000 proteins from a 250 pg K562 digest, with strong reproducibility across technical replicates. In triple proteome tests, the number of identified peptides and proteins was comparable to results from the base platform lacking the Omnitrap module. Quantitative ratios aligned closely with expected values, showing no notable discrepancies.
Conclusion
The timsOmni system, integrating MSⁿ and eXd capabilities, maintains top-tier sensitivity and quantitative precision for complex sample analysis. These findings confirm its suitability not only for in-depth structural proteomics but also for routine, high-throughput biological investigations. With its broad applicability, timsOmni stands out as a valuable asset for systems biology, biomarker identification, and functional proteomics.