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Enhanced in-Source Fragmentation Annotation Enables Novel Data Independent Acquisition and Autonomous METLIN

Jingchuan Xue1, Xavier Domingo-Almenara2, Carlos Guijas1

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Summary

Enhanced in-source fragmentation annotation (eISA) improves molecular identification by generating fragmentation patterns comparable to higher energy fragments. This method increases precursor ion intensity and identification confidence, offering a sensitive alternative to existing techniques.

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Area of Science:

  • Analytical Chemistry
  • Mass Spectrometry
  • Molecular Identification

Background:

  • Electrospray ionization (ESI) in-source fragmentation (ISF) is typically minimized, limiting its utility in molecular identification.
  • Current in-source annotation algorithms are constrained by ESI sources designed to suppress ISF.
  • Existing methods like data dependent acquisition (DDA) and data independent acquisition (DIA) with quadrupole time-of-flight mass spectrometry (QTOF-MS) have limitations in sensitivity and fragmentation pattern generation.

Purpose of the Study:

  • To develop and validate an enhanced in-source fragmentation annotation (eISA) method.
  • To generate in-source fragmentation patterns comparable to higher energy MS/MS spectra without compromising precursor ion intensity.
  • To assess eISA's performance against QTOF-MS DDA and DIA methods for molecular identification.

Main Methods:

  • Tuning ESI source conditions to enhance in-source fragmentation (ISF).
  • Developing the enhanced in-source fragmentation annotation (eISA) algorithm.
  • Validating eISA using 50 molecules and comparing results with METLIN MS/MS library, QTOF DDA, and QTOF DIA spectra.
  • Analyzing a metabolic extract from macrophages to demonstrate practical application.

Main Results:

  • eISA generated fragmentation patterns consistent with METLIN library data for over 90% of molecules.
  • eISA significantly increased precursor ion intensity compared to QTOF DDA (median 18x in negative, 210x in positive mode) and QTOF DIA (median 20% increase in negative, 80% in positive mode).
  • eISA improved molecular identification confidence in low-resolution single quadrupole mass spectrometry experiments.
  • eISA proved to be a more sensitive alternative to QTOF DIA and DDA approaches.

Conclusions:

  • Enhanced in-source fragmentation annotation (eISA) effectively generates informative fragmentation patterns.
  • eISA enhances molecular identification confidence and sensitivity across various mass spectrometry platforms.
  • This method represents a valuable advancement for untargeted LC/MS experiments and molecular discovery.