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Spectronaut is commercial software from Biognosys for analysing data-independent acquisition (DIA) mass-spectrometry proteomics data. This guide is for researchers and core-facility staff who need to know what it does, what it needs as input, how to run a library-free (directDIA) analysis, and what the free alternatives are. Menu names below come from the Spectronaut 20 manual; Biognosys lists version 21 as the current release, so wording may differ slightly in newer builds.
What Spectronaut is used for in research
Biognosys describes Spectronaut as software for the comprehensive analysis of DIA proteomics experiments, covering the whole workflow from search space and quantification to visualisation and reporting. For the acquisition background, see choosing DDA or DIA, sample prep and run QC.
Spectronaut supports two ways of identifying peptides:
- directDIA (library-free): the DIA runs are deconvoluted into pseudo-MS/MS spectra and searched with the built-in Pulsar engine against a protein FASTA file. The manual states that this works "using nothing but a FASTA file".
- Library-based: a spectral library is generated with Pulsar from DDA, DIA or hybrid data, or imported from search archives, external results or files.
Biognosys also lists a PTM site-level workflow (localisation and differential abundance of modification sites) and, from version 17, directDIA+ modes: directDIA+ (Deep) for the deepest coverage and directDIA+ (Fast) for a balance of speed and depth in analyses without PTMs.
Inputs and outputs
| Item | What Biognosys lists |
|---|---|
| DIA raw data | Thermo, SCIEX, Bruker and Waters instrument files |
| Sequence database | FASTA file(s) |
| Spectral libraries | .kit, .txt, .csv, .tsv, .xls |
| External search results | MaxQuant, Proteome Discoverer, ProteinPilot, Mascot, mzIdentML |
| Analysis file | .sne, which can be saved with or without ion traces (XICs); the version without XICs is smaller |
| Reports | Customisable reports, a PTM Site Report, differential abundance candidates and volcano plots |
Source: Biognosys Spectronaut product page and Spectronaut 20 manual (read 2026-11-08).
Install or access (official source only)
Spectronaut is licensed software. Biognosys offers licences through a quote request form (Standard, Advanced and Premium tiers, system-bound or floating) and a 30-day trial through a separate trial form, both on the official Spectronaut page. Pricing is quoted by Biognosys and is not stated here.
System requirements listed by Biognosys:
- Windows 10 or Windows Server 2016 as the minimum, Windows 11 recommended.
- Linux (Ubuntu 22.04) in command-line mode only. From version 18 the manual describes Linux support for library generation, directDIA, library-based analysis and combining .sne files.
- Recommended: 16 or more cores, 16 GB or more RAM, 2 TB or more free disk space, and .NET 8.0 or later. Cloud deployment is supported.
A free Spectronaut Viewer opens .sne files for inspection but does not analyse mass-spectrometry data; Biognosys handles access requests by email. The manual notes that .sne files can be exchanged between operating systems and viewed in the Windows Viewer.
Step-by-step: run a directDIA analysis
These steps follow section 3.4.1.2 of the Spectronaut 20 manual. You need your DIA raw files and a FASTA file for the species you studied.
- Open the Analysis Perspective (Spectronaut starts there). Add an experiment tab if you need a new one.
- Click Set up a directDIA Analysis… at the bottom left.
- Choose the run files (or copy the files to the clipboard and paste them).
- In the wizard, name the analysis.
- Choose the FASTA file(s).
- Choose the search and analysis settings schema. The manual says "BGS Factory Settings (default)" fits most projects.
- Specify the experimental set-up (conditions and replicates). This is what enables differential abundance testing.
- Optionally add a Gene Ontology annotation file, which allows GO enrichment and clustering.
- Optionally add extra DDA or DIA runs or Search Archives to build a hybrid library.
- Review the summary and click Finish.
Spectronaut then returns to the Analysis Perspective and starts the analysis. Internally it works in two steps: a spectrum-centric search builds a library from the DIA runs against your protein database, and a targeted analysis of the same runs then uses that library. If you want to keep the generated search archive, the manual points to the Reporting node of the Global Settings. Supported acquisition platforms for directDIA are Thermo Orbitrap, SCIEX TripleTOF, Bruker timsTOF Pro and Waters Synapt.
Output and how to read it
- Analysis Perspective: when the run finishes, the number of unique precursors, peptides and protein groups identified at the q-value cutoff appears at the bottom right. The default cutoff is 0.01, which the manual describes as equivalent to a 1% FDR. For what a q-value means, see Q-values and the positive false discovery rate.
- Conditions and statistics: unless you change it, Spectronaut runs an unpaired two-sample t-test between all conditions; a paired option exists. The "Is Reference" column sets the reference condition, and condition set-up can be redefined under Settings, Condition Setup.
- Post Analysis Perspective: shows identification and quantification summaries, differential abundance results, hierarchical clustering, principal component analysis and GO enrichment. The Candidates table is filtered by default to a q-value of 0.05 and an absolute log2 ratio of 0.58; fold changes are log2 ratios of averaged replicates (AVG Log2 Ratio). The same filters drive the volcano plot, and the table can be exported with Export Table….
- Report Perspective: choose a report schema, pick columns, apply filters, preview, then click Export Report… at the bottom left. Custom schemas can be saved and reused.
To understand how identifications are scored against a database in general, see protein identification by mass spectrometry.
Troubleshooting
The manual gives these practical rules:
- Run analyses on local disks, not network drives or virtual machines; a dropped network connection can abort an analysis.
- To limit CPU use, use Settings Perspective > Global > General > Override CPU Affinity.
- Move the default location of Local Search Archives and temporary directories to disks with enough free space.
- Errors are shown in red. The log in the Analysis or Library Perspective can be saved as text, and the About perspective has a "Show error logs" option. Biognosys also runs a helpdesk at help2.biognosys.com.
Free alternatives
| Tool | Cost and licence (per its own pages) | Inputs and notes |
|---|---|---|
| Spectronaut Viewer | Free | Inspects existing .sne files only; no mass-spectrometry analysis |
| DIA-NN (Academia edition) | Download offered for non-profit academic research, with limited functionality. A separate Enterprise edition for industry and academia is obtained through Aptila Biotech or Thermo Fisher. | Reads Thermo .raw, Bruker .d, Sciex .wiff (via ProteoWizard DLLs), .mzML and .dia; has a GUI and a command-line interface |
DIA-NN details are from the project README at github.com/vdemichev/DiaNN (read 2026-11-08); check the licence terms in the release you download.
FAQ
Is Spectronaut free?
No. Spectronaut is licensed by Biognosys, with a 30-day trial available on request. Only the Spectronaut Viewer for .sne files is free.
Do I need a spectral library?
No. directDIA builds a library from your own DIA runs using a FASTA file. A library-based analysis is the alternative if you already have one.
Which operating systems does it run on?
Windows 10 or Server 2016 and later (Windows 11 recommended), plus Ubuntu 22.04 through the command line only, according to Biognosys.
What does the 0.01 q-value mean?
It is the default identification cutoff, described in the manual as the equivalent of a 1% FDR. See the proteomics overview for the wider field.
Sources
- Biognosys, Spectronaut product page (read 2026-11-08).
- Biognosys, Spectronaut 20 Manual, sections 1.4, 1.5, 3.4, 3.5, 3.6 (read 2026-11-08).
- DIA-NN project README, github.com/vdemichev/DiaNN (read 2026-11-08).
