Creating and Using Free Public and User Libraries in NIST26 Chromatogram and Lib Search

Significance of the topic

The use of spectral libraries is central to routine and research mass spectrometry workflows for identification of small molecules and metabolites. Curated commercial libraries (notably NIST) provide the best-balanced accuracy and coverage, but open-access public libraries (MoNA, MS-DIAL and others) and bespoke user libraries expand chemical breadth, offer up-to-date entries, and enable laboratory-specific curation. Efficient combination and correct handling of these resources within NIST26 Search Software (including integrated deconvolution and library searching) improves identification depth and confidence for both EI GC–MS and LC–MS/MS applications.

Objectives and overview of the material

This handout/video documents practical steps and recommendations for creating, combining and using free public and user spectral libraries in NIST26 Chromatogram and Library Search. Key aims are to summarize available public resources, provide suggested search tolerances for different match modes (high‑ and low‑resolution data), highlight pitfalls (e.g., entries without structures), and recommend a workflow that leverages the curated NIST26 tandem library together with open libraries to maximize identification coverage.

Methodology

The approach described consists of assembling open-access LC–MS/MS libraries in NIST-compatible format, testing them in different NIST26 search modes, and adjusting tolerances to capture both high-resolution and legacy low-resolution spectra. Practical recommendations include combining MoNA LC–MS/MS experimental downloads and MS‑DIAL exports, verifying structure availability for hits, and avoiding overly restrictive filtering (for example, disabling "Best Matching Only" when needing duplicate hits with structures). Several NIST26 search modes were emphasized: Identity MS/MS, Similarity MS/MS Hybrid (with precursor-in-spectrum option) and Identity HiRes NoPrecursor.

Instrumentation used

Although no specific mass spectrometer model is required, the workflow relies on the following software resources and libraries:

• NIST26 Search Software with integrated deconvolution and library searching
• NIST 2026 tandem (MS/MS) library (commercial, curated)
• MoNA LC–MS/MS public experimental download
• MS‑DIAL public MS/MS exports (positive and negative ion sets)
• User-created NIST-format libraries derived from public resources

Main results and discussion

Key quantitative and qualitative findings from the presented material:

• Combined public libraries: MoNA (153,635 spectra; ~100k positive, ~50k negative), MS‑DIAL positive (322,191) and MS‑DIAL negative (45,483) together yield ~521,309 MS/MS spectra, with ~150,404 entries having associated structures.
• Approximately 450,000–550,000 spectra in this merged set report exact fragment masses to three or more decimal places, representing roughly 30,000 unique compounds.
• NIST26 tandem library is substantially larger and more curated: ~3.2 million spectra across about 68,635 compounds, offering deeper coverage and more consistent identification performance.
• Recommended strategy: use NIST26 as the primary, curated library and augment it with MoNA and MS‑DIAL converted to NIST format to extend coverage. This combines the high reliability of NIST curation with the breadth of open libraries.
• Search tolerance behavior: for high-resolution searches, product ion tolerances such as ±40 ppm perform well. Public libraries contain many low-resolution spectra, so testing broader tolerances for product and precursor ions may be necessary. Identity HiRes NoPrecursor searches can be run with product ion ±40 ppm; for low‑resolution entries, tolerances up to several hundred ppm may be needed. The presenter noted inconsistent behavior with tolerances >479 ppm in some modes and advised caution; NIST high-resolution library (hr_msms_nist) is tolerant of broader settings.
• Entries without structures: many public-library hits lack structural information. When investigating Chromatogram Search results, avoid using "Best Matching Only" if you need to surface duplicate records that include structures. Use right-click library searches to inspect alternative entries.
• Creating user libraries: converting current public resources into NIST format and building a unique, curated user library is highly recommended. A prior heuristic of prefixing library names with "lr_" to force low‑resolution handling did not reliably trigger NIST low‑resolution corrections—explicit tolerance settings remain necessary.

Benefits and practical applications

The combined use of NIST26 and compatible public libraries provides these practical benefits:

• Broader compound coverage for untargeted and suspect screening workflows, especially in environmental, forensic, metabolomics and food analysis contexts.
• Cost-effective extension of a laboratory’s spectral resources by incorporating free public spectra alongside a relatively inexpensive NIST26 purchase.
• Improved identification confidence by pairing NIST’s curated core with the diverse experimental spectra from MoNA and MS‑DIAL.
• Ability to tailor a user library to local sample types, instrument platforms and QA/QC practices, improving routine identification rates.

Future trends and potential applications

Expected developments and opportunities include:

• Continued growth and curation of large commercial libraries (e.g., NIST) with deeper coverage and improved metadata, facilitating more reliable automated identifications.
• Better integration of community‑curated open libraries into commercial software via standardized conversion tools and improved provenance/structure annotation.
• Enhanced deconvolution and library‑search algorithms in platforms like NIST26 to exploit broad hybrid libraries, increasing hit quality for coeluting compounds and low‑abundance signals.
• Machine-learning–assisted scoring and spectral prediction to bridge gaps where experimental spectra are absent, and to prioritize likely structural candidates from public libraries that lack assigned structures.
• Automation of routine library conversion and update processes so labs can keep local user libraries current with community resources.

Conclusion

Combining a curated commercial backbone (NIST26 tandem library) with converted public libraries (MoNA, MS‑DIAL) delivers a pragmatic balance of accuracy and breadth for LC–MS/MS identification tasks. Properly tuned search tolerances and careful handling of entries without structures are essential. Building and maintaining a bespoke user library remains a high‑value practice for any lab aiming to optimize identification rates and reproducibility. NIST26’s integrated deconvolution and library search capabilities amplify the benefit of merging curated and open resources.

References

• James Little. Creating and Using Free Public and User Libraries in NIST26 Chromatogram and Lib Search. Video/Handout. Mass Spec Interpretation Services, April 25, 2026.
• MoNA (MassBank of North America) public LC–MS/MS datasets, public repository releases (May 2025).
• MS‑DIAL public MS/MS exports (2025–2026 releases).
• NIST Tandem MS Library (2026 release) documentation and NIST26 Search Software release notes.