Data Processing for NIST MSMS Search Using Thermo Freestyle Software (My Favorite Approach)

This webinar series demonstrates the use of the Thermo Freestyle 1.8 SP2 QF1 software for processing LCMS product-ion mass spectra  with the NIST 23 Mass Spectral Search Program (v.3.0).  Spectra are easily exported from Freestyle to NIST Search for component identifications utilizing the NIST MSMS libraries and any other available user, crowd-sourced, and commercial MSMS libraries.  Freestyle is a very convenient software package for processing data obtained with the their Orbitrap LCMS instruments.  In one run both the MS1 and MS2 spectra are obtained in both positive and negative ion modes.  The Diode arrary and UV channels can also be easily displayed for targeting the desired species noted in LC analyses.

Handout for whole series

• Part 1: My Favorite Processing Mode in Freestyle
• Part 1A: Using "Nearby Precursor" Enhanced Processing Mode in FreeStyle
• Part 2: Alternate Processing Modes in Freestyle
• Part 3: Setup of Basic Settings in Freestyle and NIST Search
• Part 4: Different NIST MSMS Searches Demonstrated
• Part 5: Elemental Processing within Thermo Freestyle

Part 1: Data Processing for NIST MSMS Search Using Thermo Freestyle Software (My Favorite Approach)

This guide outlines my preferred method for processing data in Thermo Freestyle software for NIST MSMS searches. These steps streamline data parsing and ensure accurate mass spectral interpretation for LC-MS and MSMS analyses.

Equipment & Software

• Thermo Orbitrap MSMS
• Thermo Freestyle Software
• NIST 2023 Tandem Library

Orbitrap Data One Acquisition

• LC-MS and MSMS Analyses
• Non-targeted acquisition of MSMS
• Positive and Negative Mode
• Photo Detector Array (PDA) and UV-VIS Analyses
• Example compounds:
  • Uvinul 3000 (MW 214)
  • Tinuvin 234 (MW 447)

James Little: NIST 2023 Tandem Library Great Resource for Unknown Identifications

My Favorite Data Processing Workflow

• shared with me by colleague

Step 1: Setting Up Freestyle Windows

To optimize the workspace for data processing:

1. Open three windows at the bottom of Freestyle:
   • Top window: Positive ion MS1
   • Second window: Negative ion MS2
   • Bottom window: UV or PDA trace
2. Save this layout in Workspace Options/Layouts to ensure automatic parsing when opening new files.
3. Add the Nearby Precursors feature to simplify MSMS selection (except for averaged MS spectra).

James Little: Step 1 - Setting Up Freestyle Windows

Step 2: Adjusting the Window Layout

1. Use Select Filter to:
   • Change the top window to positive ion
   • Change the second window to negative ion
   • Choose PDA or UV for the bottom window
2. The spectrum window will display the selected MS/MS data for further processing.

James Little: Step 2 - Adjusting the Window Layout

Step 3: Processing MSMS Data

1. Left-click in the top box for the positive ion chromatogram.
2. Right-click to average the spectrum in the Spectrum box.
3. Subtract background if necessary before averaging.
4. Populate the Avg Range box and select Range 1.
5. Go to Positive Ion Chromatogram, specify the range, and confirm.
6. Avoid averaging MS2 spectra when exporting to NIST search, as it will convert accurate mass to nominal mass.

James Little: Step 3 - Processing MSMS Data

James Little: Step 3 - Processing MSMS Data

James Little: Step 3 - Processing MSMS Data

Step 4: Selecting an MS2 Spectrum for NIST Search

1. Identify an m/z value of interest (e.g., m/z 215.0702).
2. Apply Chromatogram range filter to isolate the target ion.
3. The MS2 spectrum will replace the MS1 chromatogram in the top window.
4. Click on the peak of interest to generate an MS2 spectrum.
5. Repeat the process for Negative Ion MS2 (MSMS) spectrum.
6. Send spectra to NIST MS search for compound identification.

James Little: Step 4 - Selecting an MS2 Spectrum for NIST Search

Conclusion

Using this workflow, Thermo Freestyle software efficiently processes LC-MS/MS data for NIST searches, ensuring accurate identification of unknown compounds. By saving custom layouts and avoiding averaged MS2 spectra, this method maximizes precision and efficiency.