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Mass Spectrometry: Molecular Fragmentation Overview01:20

Mass Spectrometry: Molecular Fragmentation Overview

The ionization of a molecule into a molecular ion inside the mass spectrometer causes instability in the molecule's structure due to the loss of an electron. This eventually leads to the fragmentation or breaking of some bonds in the molecule. The fragmentation occurs predominantly at specific bonds to yield relatively stable fragments.
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Tandem mass spectrometry is a technique that uses multiple mass analyzers in series to obtain a higher selectivity and reduce chemical noise during analyte detection. Instruments with multiple analyzers separated by an interaction cell enable secondary fragmentation and selected study of the fragment ions.Secondary fragmentations occur in the interaction cell and can be induced by various factors. Fragmentation induced by collision with inert gases, such as N2, Ar, He, etc., is called...
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  1. Home
  3. Imfrag: A Tool To Recognize In-source Fragmentation In Ion Mobility-enabled Data-independent Acquisition Workflows.
  1. Home
  3. Imfrag: A Tool To Recognize In-source Fragmentation In Ion Mobility-enabled Data-independent Acquisition Workflows.

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Navigating the Mass Spectrometry-Based Proteomic Data Using Free Computational Tools
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Published on: August 19, 2025

IMFrag: A Tool to Recognize In-Source Fragmentation in Ion Mobility-Enabled Data-Independent Acquisition Workflows.

Ryan Nguyen1, Anvitaa Anandkumar1, Griffin Rangel1

  • 1Department of Medicinal Chemistry, University of Washington, Seattle, Washington 98195, United States.

Analytical Chemistry
|June 6, 2026

View abstract on PubMed

Summary
This summary is machine-generated.

In untargeted metabolomics, in-source fragmentation (ISF) causes misidentification. IMFrag, using ion mobility-mass spectrometry (IM-MS), differentiates ISF from true fragments, improving compound identification in complex samples.

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Area of Science:

  • Analytical Chemistry
  • Biochemistry
  • Computational Chemistry

Background:

  • High-resolution mass spectrometry is vital for untargeted metabolomics and exposomics.
  • In-source fragmentation (ISF) poses a significant challenge, leading to compound misidentification.
  • Current methods struggle to distinguish ISF from genuine MS/MS fragments, hindering accurate analysis.

Purpose of the Study:

  • To develop a computational tool, IMFrag, for differentiating in-source fragments from true product ions using ion mobility-mass spectrometry (IM-MS).
  • To enhance the accuracy of compound identification in untargeted metabolomics and exposomics studies.
  • To leverage structural information from IM-MS to improve MS/MS spectral library matching.

Main Methods:

  • Development of IMFrag, a Jupyter notebook-based tool integrating IM-MS data.
  • Utilizing ion mobility signatures to distinguish fragments generated at different stages of the mass spectrometry process.
  • Application of IM-enabled data-independent acquisition (IM-DIA) for analyzing l-tryptophan and diverse small molecules.
  • Validation using complex biological matrices, including human plasma samples.

    • Main Results:

    • IMFrag successfully differentiates ISF from collision-induced dissociation (CID) fragments based on distinct ion mobility signatures.
    • Distinct mobility patterns were observed for identical fragment ions originating from different fragmentation events.
    • IM-DIA provided additional structural insights, such as dimer formation and protonation sites, not evident in standard LC-MS.
    • The tool demonstrated utility in resolving ambiguities in complex biological samples, like coeluting isobaric compounds.

    Conclusions:

    • IMFrag provides an accessible framework for analyzing MS¹ and post-IM MS² data in untargeted studies.
    • The integration of IM-MS data significantly improves the reliability of compound identification by mitigating ISF.
    • This approach enhances untargeted analysis pipelines and aids in the development of robust MS/MS spectral libraries.