Spectronaut: A Groundbreaking Increase in Identifications

Spectronaut® 17: A Groundbreaking Increase in Identifications

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Spectronaut® 17 brings the ultimate solution to library-free DIA analysis with directDIA+™. Our novel patent-pending algorithm works by expanding the spectrum-centric search in directDIA™ with an improved in-silico predicted search space. The result is an unprecedented proteome coverage with no compromises in quantification quality.



“With directDIA+, Spectronaut 17 offers a completely novel way of analyzing DIA proteomics data. With this new workflow, our users will experience the deepest proteome coverage to date with the least possible resources. We are thrilled to offer this tool to our customers.”


Lukas Reiter, Ph.D., CTO of Biognosys

“For most of our DIA projects, Spectronaut’s directDIA is our standard analysis workflow. We love that the software is click-and-play and that you don’t need to spend extra time and samples optimizing a project-specific library. Now, with directDIA+ in Spectronaut 17, we don’t have to make any compromises: we get a proteome coverage that is comparable, if not better than with a deep dedicated library.”


Prof. Dr. Jesper Olsen, CPR University of Copenhagen

Feature Overview

  • Unmatched proteome coverage: The directDIA+ algorithm significantly improves identifications across all major DIA-compatible instruments




  • Results you can trust: Robust false discovery control validated with several datasets


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Unmatched Proteome Coverage Across Instruments

With the new directDIA+ workflow Spectronaut 17 achieves unprecedented proteome depth from a library-free approach across all major DIA-compatible instruments.


Spectronaut 17 identifies more protein groups on timsTOF, Orbitrap, and ZenoTOF with a median improvement of 21% on timsTOF, 10% on Orbitrap, and 45% on ZenoTOF (not pictured here).

More Quantitative Proteins, No Library Needed

The greatly improved identification rate with directDIA+ also translates to more quantitative proteins than ever before.


We measured mixed proteome samples of four species spiked in different amounts (C. elegans, E. coli, H. sapiens and S. cerevisiae). As shown in the left panel, Spectronaut 17 does an excellent job at quantifying all four species despite identifying many more proteins.


Regulation analysis was performed using an unpaired t-test of protein quantities. All the regulated proteins were sorted by p-value and categorized as true and false positives based on the ground truth pipetting. Spectronaut 17 recovered 13% more true candidates than version 16 at 5% FDR.

Benefit from the Best Library-free Workflow for PTM Studies

Spectronaut 17 makes PTM studies easy. Here we show correct localization of phospho peptides by Spectronaut in a benchmark experiment.


Spectronaut 17 in library-free directDIA+ mode shows excellent performance when true and false localization rates are compared using JPT, a set of synthetic peptides with known localization benchmarked in different concentrations of a yeast background matrix1.

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