Data Processing Using Thermo Freestyle Software with NIST MSMS Search for Compound Identifications (Elemental Composition with Freestyle)

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 5: Data Processing for NIST MSMS Search using Thermo Freestyle Software (Elemental Composition with Freestyle)

Equipment & Software

• Thermo Orbitrap MSMS
• Thermo Freestyle Software
• NIST MSMS Search

Elemental composition analysis is a crucial step in mass spectrometry-based compound identification. Using Thermo Orbitrap MSMS in combination with the Thermo Freestyle software and NIST MSMS Search, researchers can achieve high-precision molecular formula determinations with excellent isotope ratio measurements. This article provides a step-by-step guide on utilizing these tools for accurate compound identification.

Benefits of Elemental Composition Analysis

• Accurate m/z Measurements: Ensures precise molecular formula determinations.
• Excellent Isotope Ratios: Helps refine possible elemental compositions.
• Minimized Results List: Using both accurate m/z values and isotope ratios significantly reduces the number of potential matches.

Step-by-Step Guide: Using Thermo Freestyle for Elemental Composition

• Open the Elemental Tab in the Thermo Freestyle software.

James Little: Elemental Tab

• Set Up Elements and Element Ranges based on the expected composition of the sample.
• Activate "Pick Peak" Mode to select the ion of interest.
• Click on an Ion Mass Value in the MS1 Spectrum window.
• Automatic Transfer of Mass Value: The selected value will appear in the “mass” box in the Elemental window.
• Click "Calculate" to generate potential elemental compositions.
• Review Results in the window below the spectrum display.
• Analyze Single or Average Spectra: The method applies to both single and averaged mass spectra.

James Little: Step-by-Step Guide Using Thermo Freestyle for Elemental Composition

Example Compound: Tinuvin 234

Tinuvin 234 is a widely used light stabilizer. Its analysis using the Thermo Orbitrap MSMS and NIST MSMS Search demonstrates the effectiveness of this workflow in compound identification.

James Little:Tinuvin 234

Conclusion

Leveraging Thermo Freestyle software alongside high-resolution mass spectrometry enhances the accuracy and reliability of elemental composition determination. By refining mass selection and isotope ratio analysis, researchers can significantly reduce false-positive identifications and improve confidence in their results.