Features an integrated database search engine called Pulsar enabling spectral-library free DIA workflow - directDIA™
Supports spectral-library-free workflow (directDIA™) and targeted analysis of the data using spectral libraries (Hyper Reaction Monitoring – HRM™). More information available in the Spectronaut™ Pulsar Brochure (download here).
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Spectronaut™ Pulsar is specifically developed for the analysis of data independent acquisition (DIA) measurements. Spectronaut™ Pulsar analyzes the acquired signals with a spectral-library-free workflow, directDIA™, or with a targeted analysis of the data using spectral libraries (Hyper Reaction Monitoring – HRM™). It features Pulsar, Biognosys’ proprietary database search engine, which supports both workflows and eliminates the need for external search engines. Contact us for a FREE TRIAL!
HRM™ workflow is supported for all instruments listed above. DirectDIA™ worklow is supported for Thermo Scientific™ instruments.
Spectronaut™ Pulsar analyzes raw data from a large variety of
Methods should acquire MS1 and MS2 or MS2 only scans. Cycle time of the method should be in the range of 1-3 seconds depending on your average peak width. Gas phase fractionation is supported.
Minimum: Windows 7 x64, CPU Intel ® Core™ CPU 2.7 GHz (quad core), HDD 200 GB free space, Memory 8 GB, Software .NET 4.5.
Recommended: Windows 7 x64 or higher, CPU Intel Core i7 4770, 3.4 GHz (octa core) or more, HDD 500 GB free space (SSD), Memory 16 GB or more, Software .NET 4.5 or higher.
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If you refer to Spectronaut™ algorithms in your research please cite:
Reiter L, Rinner O, Picotti P, Hüttenhain R, Beck M, Brusniak MY, Hengartner MO, Aebersold R. mProphet: automated data processing and statistical validation for large-scale SRM experiments. Nat Methods. 2011;8(5):430-5.
If you refer to the software package please cite:
Bruderer R, Bernhardt OM, Gandhi T, Miladinovic SM, Cheng LY, Messner S, et al. Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen treated 3D liver microtissues. Mol Cell Proteomics. 2015 Feb 27. pii: mcp.M114.044305.
FASTA of iRT peptides for shotgun search
Skyline Schema for exporting Spectronaut compatible libraries - simply add this schema to Skyline's report list
Spectral library generation using Proteome Discoverer 1.4 (HEK293 cell line sample)
Proteome Discoverer 1.4 Search Files (zip, 1.3 GB) for spectral library generation
Spectral library generation using MaxQuant 1.5 (HEK293 cell line sample)
MaxQuant Search Files (zip, 386 MB) for spectral library generation
HEK293 demo data set
HEK293 demo data set
HEK cell line sample
Window sizes (txt file) - this file is needed only when analyzing the data with software other than Spectronaut™ Pulsar
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What is the difference between directDIA™ and HRM workflows?
DirectDIA™ and HRM are data independent acquisition (DIA) based workflows that provide comprehensive proteome coverage by multiplexing thousands of proteins. DirectDIA™ is a simple workflow that doesn’t require additional runs for spectral library generation. In HRM additional DDA or DIA runs are needed for spectral library generation to produce the highest possible number of identified and quantified peptides and proteins.
What is Pulsar?
Pulsar is Biognosys’ proprietary database search engine that is integrated into Spectronaut™. This search engine implements a dynamic PSM stratification strategy to maximize identifications in large data sets and to control FDR on PSM subsets such as modified peptides.
Can I use external database search engines with Spectronaut™ Pulsar?
Yes, Spectronaut™ Pulsar supports external search engines MaxQuant, Mascot, Proteome Discoverer and ProteinPilot in addition to Pulsar for HRM experiments. However, directDIA™ workflow is only possible with the use of integrated Pulsar search engine.
Is Spectronaut applying normalization and if so what strategy is used?
Per default Spectronaut normalizes the data. The two underlying assumptions are that there are on average no proteins that change in abundance (1) and if there are proteins that are changing then there is the same number of proteins upregulated vs downregulated (2).
What does the Q-value stand for and how is it different from the p-Value?
The Q-value is the minimum false discovery rate at which the test may be called significant. As the definition implies it gives a direct measure of the FDR of the identification and quantification.
What is the default Q-value cut off used by Spectronaut?
For peptide identification the cut off value is set at Q-value<0.01 for precursor identification. This means that among all true identified precursors there is 1% false positive identifications.
For the statistical testing of differentially abundant proteins the Q-value is set at Q-value<0.05.
How is the Q-value for peptide identification calculated?
In total more than 12 different measurements are used to calculate the Q-value for a given peptide. Among the values factoring into the equation are iRT accuracy, relative fragment ion intensities many more.
How are the differential abundant candidates in the Post Analysis Perspective calculated?
To determine the proteins differentially abundant between different conditions Spectronaut is performing a one sample t-test on the log ratios of all features between all conditions. For the protein quantities the average precursor intensities are used per default.
I do not like the default settings where can I change them?
The Biognosys factory settings or also called default settings have proven to be suitable for the majority of the experimental set ups. Yet, sometimes individual settings are necessary. To perform data analysis using user defined settings there are two options available. Option 1: the settings are modified in the Review Perspective when setting up the experiment. Option 2: In the Settings Perspective the settings are modified and stored in a separate settings schema.
I want to subject the data generated in Spectronaut to third party analysis tools how can I do this?
The Report Perspective is design in a way that all accessible information can be exported. For certain downstream analysis tools a reporting schema can be created based on the information needed. If you need help establishing a schema do not hesitate to contact us at email@example.com
Can I use other peptides than the iRT peptides for QC monitoring?
Yes, in the Prepare Perspective the library can be used to generate a pool of peptides suitable for QC. This additional panel can be used in the QC Perspective.
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