Processing MS/MS Data in NIST26 Chromatogram Window
- Photo: James Little: Processing MS/MS Data in NIST26 Chromatogram Window
- Video: james little: Processing MS/MS Data in NIST26 Chromatogram Window
The new NIST26 release expands LC-MS/MS data processing with integrated chromatogram analysis and MS/MS library searching. Within the Chromatogram Window, users can load LC-MS/MS data, configure processing parameters, search spectral libraries, filter results, review identified components, and export selected results for further evaluation.
This practical guide is based on James Little’s Processing MS/MS Data in NIST26 Chromatogram Window material from Mass Spec Interpretation Services. It walks through the fundamental workflow for processing MS/MS data in the NIST26 Chromatogram Window, from selecting the appropriate processing mode and input file to library searching, reviewing candidate identifications, and exporting results.
Before You Begin: Navigating the Chromatogram Window
The workflow assumes that users are already familiar with configuring and navigating the NIST26 Chromatogram Window. For new users, the author recommends first reviewing the separate material dedicated to configuring and navigating this environment.
NIST26 also provides extensive integrated help. Pressing F1 while working in the Chromatogram Window opens documentation and practical tips for processing tandem MS data. The presentation emphasizes that this help resource contains useful information for understanding individual functions and settings.
1. Set Processing to Tandem Mode
If both EI and tandem NIST software are installed, the first useful step is to specify the type of spectra being processed.
In the Chromatogram Window, select: File → Select Spectrum Type → Tandem
Selecting Tandem mode simplifies menu navigation and ensures that functions relevant to MS/MS data processing are readily available.
2. Prepare Your Data in mzML Format
For the workflow described in the presentation, NIST26 processes mzML files. Data stored in proprietary instrument formats therefore need to be converted before processing.
The presentation recommends using ProteoWizard/msconvert for file conversion.
Once the mzML file is available, processing can be started through: File → Analyze MS/MS Input File
Select the appropriate mzML file and configure the required processing parameters.
3. Set the Mass Tolerance
Mass tolerance is one of the important parameters used during data processing and spectral matching.
The presentation identifies:
- 20 ppm
- 40 ppm
as reasonable tolerance values.
The appropriate setting should ultimately reflect the characteristics of the dataset and instrumentation. These values should therefore be regarded as practical starting points for the demonstrated workflow rather than universal settings for every LC-MS/MS analysis.
4. Select Libraries for Searching
One of the useful features of NIST26 is the ability to search multiple libraries simultaneously.
For the type of analysis demonstrated, James Little identifies hr_msms_nist as the preferred library. Depending on the application, users can also work with APCI and low-resolution libraries or free crowd-sourced libraries.
Searching several relevant libraries can broaden the identification space, particularly when investigating complex LC-MS/MS datasets.
5. Examine the Initial Results
After processing is complete, the results are displayed directly within the Chromatogram Window. The workspace combines several types of information, including:
- the chromatogram
- a list of detected components and search results
- information about library hits
- comparison of experimental and library spectra
- proposed compound identity and structural information
This layout makes it possible to move quickly from a chromatographic feature to its potential identification and visually evaluate the quality of the spectral match.
Abundance can also be displayed using either a linear or logarithmic scale. A logarithmic view can be particularly helpful when the chromatogram contains both highly abundant signals and much weaker components. Pages 6 and 7 of the presentation illustrate the difference between these two views.
6. Save Your Configuration
Once the desired program parameters have been established across the relevant windows, including the Chromatogram Window, it is useful to save the configuration.
NIST allows configurations to be saved and restored as *.ini files.
This can be particularly valuable when a laboratory uses different processing configurations for different datasets, libraries, or analytical workflows. Instead of manually recreating the settings each time, users can restore a previously saved configuration.
According to the presentation, the Save Configuration option is available from the menu in the Lib Search tab.
7. Filter the Results
Initial processing can generate a large number of potential identifications. NIST26 therefore provides several options for filtering and simplifying the results.
Functions available from the upper area of the Chromatogram Window include:
- Best Hits
- Only IDs
- Bkgd
- Filter Scores
Changing these filters affects the displayed hit list and the identifications shown in the chromatogram. This allows users to progressively narrow the results and concentrate on components that are most relevant to the analytical task.
Be Careful with the Background Filter
The presentation specifically highlights the Bkgd setting.
According to the material, NIST suggests a background setting of approximately 75%, and this is normally useful. However, the author observed a few instances in which applying the background filter removed major peaks despite their good library search scores.
These peaks had an XIC value of 0.
If an important candidate disappears after applying background filtering, the settings should therefore be reviewed. For these particular peaks, the presentation notes that Bkgd = Any was required.
8. Use Filter Scores with Care
The Filter Scores function provides another way to remove lower-quality spectra from the displayed results.
According to the NIST help information cited in the presentation, filtering is based on five quality factors:
- background
- purity
- max2min
- signal-to-noise
- isotope analysis
The filter can be configured to display only identifications above a specified minimum score.
The procedure presented is:
- Turn on Filter Scores above the chromatogram.
- Enter the desired minimum score.
- Select Fixed in the corresponding selection box.
- Only identifications above the specified score will remain visible.
The presentation gives 900 as an example setting for obtaining a clear view of the TIC.
The author also notes that the practical interpretation of this feature is not necessarily straightforward and recommends experimenting with it to determine whether it is useful for a particular workflow.
9. Sort Results and Create a List of Components of Interest
The results table offers considerable flexibility for organizing potential identifications.
Clicking a column header allows the results to be sorted according to the selected parameter. Multiple entries can be selected using Shift or Ctrl together with the left mouse button.
Selected entries can then be sent to a smaller list containing only components of interest.
The presentation emphasizes that there are many possible approaches to organizing the results. Rather than imposing a single rigid workflow, NIST26 allows users to adapt result selection and review to their specific analytical requirements.
10. Manually Review Candidate Identifications
Automated library searching should not necessarily be treated as the final step in compound identification. Once a list of relevant components has been created, the presentation recommends reviewing the candidates individually.
A practical approach is to:
- Sort the results by RT (retention time).
- Left-click the first hit to highlight it.
- Use the up and down arrow keys to move through the list.
- Carefully inspect the spectral comparison for every candidate.
- Decide whether each identification should be included in the final report.
An important tool for this review is the butterfly display in the upper-right portion of the workspace. It provides a visual comparison of the measured and reference spectra.
Combining automated search scores with expert examination of the spectral match provides a more considered approach to reviewing proposed identifications.
11. Export Selected Results to Excel
Once the relevant identifications have been reviewed, selected results can easily be transferred into a spreadsheet.
Highlight the desired entries, right-click, and choose:
Copy Selected Hits to Clipboard
The copied information can then be pasted directly into Microsoft Excel.
This provides a convenient way to create a customized results table for further evaluation, documentation, or preparation of an analytical report. The workflow is illustrated on page 13 of the presentation.
12. Perform a More Detailed Review with the XIC Browser
For candidate identifications requiring further investigation, the corresponding spectrum can be sent to the XIC Browser/XIC Analyzer.
From the selected result, right-click and choose: Send To → XIC Analyzer
This opens a more detailed view of the MS/MS data.
The example on page 14 demonstrates how the isotope pattern can support an identification involving two chlorine atoms. The XIC Browser also provides MS1 information underlying the MS2 spectrum, adding another layer of information for evaluating a candidate.
As with the main Chromatogram Window, users can press F1 or access Help for more detailed information about the XIC Browser and its functions.
Recommended NIST26 Workflow
The overall procedure can be summarized as:
mzML data → Tandem mode → mass tolerance settings → MS/MS library selection → data processing → result filtering → selection of relevant components → manual spectral review → XIC review → export of results
A key advantage of the NIST26 Chromatogram Window is its flexibility. Users do not have to accept every automatically proposed identification. Instead, results can be filtered, sorted, organized into custom lists, and individually reviewed before being included in the final report.
Conclusion
The NIST26 Chromatogram Window provides a more integrated environment for processing LC-MS/MS data and performing spectral library searches. The workflow presented by James Little begins with mzML data and appropriate Tandem mode configuration, followed by library selection, processing, filtering, and systematic review of candidate identifications.
An important principle is not to rely exclusively on automated scores. Filters can help transform a large results set into a manageable list of candidates, but final evaluation should also consider spectral comparisons, chromatographic information, and, where appropriate, additional examination in the XIC Browser.
The ability to save processing configurations and export selected results to Excel further supports the development of practical and repeatable workflows for routine LC-MS/MS data processing.
