Proteomics researchers are gaining a powerful new ally in their quest for deeper, faster, and more precise protein analysis. Recent collaborative work by Biognosys Group and Bruker demonstrates that the combination of diagonal-PASEF acquisition on the timsTOF HT platform with Spectronaut’s advanced data analysis capabilities is redefining what’s possible in discovery proteomics.
By leveraging fast ion mobility ramping and efficient diagonal quadrupole scanning, diagonal-PASEF achieves an exceptionally high cycle speed, allowing for the oversampling of chromatographic peaks. Through summation of scans for optimal peak sampling, the method delivers higher signal-to-noise ratios and significantly improved identification and quantification, enabling peptides and proteins to be measured with unprecedented precision.
Published in Molecular & Cellular Proteomics, the study led by Roland Bruderer, Vice President R&D, and Tejas Gandhi, Vice President Bioinformatics at Biognosys Group, showed that diagonal-PASEF combined with retention-time summation (RTsum) consistently outperformed traditional dia-PASEF approaches. On HeLa cell line samples, the workflow increased peptide and protein identifications while enhancing quantitative precision at both peptide and protein levels. Controlled quantitative experiments further showed that diagonal-PASEF identified more true positive candidates for differentially abundant peptides and proteins at stringent error thresholds, underlining its value for complex proteomics studies.
Comparison of peptides and percentage of peptides identified using dia-PASEF and diagonal-PASEF for short gradients combined with Spectronaut®. (© Below, Bernhardt, Kaspar-Schönefeld et al. Molecular & Cellular Proteomics, 2025)
The flexibility of diagonal-PASEF is another major advantage. With slice configurations ranging from one to twelve, researchers can tailor cycle times and sampling strategies to match sample load and experimental goals. Low sample amounts benefit from faster, fewer-slice methods, while higher loads can exploit longer cycles with more slices—all fully supported by Spectronaut® 20 with minimal setup.
According to the authors, “Optimized diagonal-PASEF and Spectronaut® 20 set a new benchmark for speed, depth, and precision in proteomics.”
These advances establish diagonal-PASEF as a competitive, future-ready approach to DIA. In combination with Spectronaut, diagonal-PASEF workflows can be seamlessly integrated into existing analysis pipelines, delivering enhanced protein identification and quantification and driving transformative discoveries in biology and medicine.
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Reference: Enhanced Identifications and Quantification through Retention Time Down-Sampling in Fast-Cycling diagonal-PASEF Methods, Below, Christopher R. et al., Molecular & Cellular Proteomics, 101480. DOI: 10.1016/j.mcpro.2025.101480
