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Directly matching isotopic distributions in top-down mass spectrometry (TDMS) improves analysis of large proteins like monoclonal antibodies. This novel isotope fitting strategy enhances proteoform identification and sequence coverage.

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Proteomics

Background:

  • Top-down mass spectrometry (TDMS) analyzes intact proteins and antibodies for modifications and variants without enzymatic digestion.
  • Interpreting TDMS spectra for large molecules like monoclonal antibodies is challenging due to numerous fragment ions.
  • Current methods often rely on mass deconvolution, which can suffer from poor sensitivity and overlapping distributions.

Purpose of the Study:

  • To introduce and evaluate an alternative spectral matching approach for TDMS analysis of large proteins.
  • To overcome the limitations of mass deconvolution in interpreting complex TDMS spectra.
  • To enhance the identification and characterization of intact biotherapeutics and other large biomolecules.

Main Methods:

  • Developed a workflow for direct matching of theoretical proteoform isotopic distributions to TDMS spectra.
  • Applied the isotope fitting strategy to TDMS data of an intact NIST monoclonal antibody across various fragmentation modes.
  • Compared the performance of isotope fitting against traditional mass deconvolution methods.

Main Results:

  • The isotope fitting strategy significantly increased sequence coverage for both light and heavy chains (>3-fold).
  • This method proved effective in identifying large fragments, including those from the hinge region, which are difficult to analyze.
  • The approach avoids sensitivity issues and overlapping distribution problems associated with mass deconvolution.

Conclusions:

  • Direct isotope fitting is a powerful alternative to mass deconvolution for TDMS analysis of large molecules.
  • This advancement improves the characterization of intact biotherapeutics and expands the utility of top-down mass spectrometry.
  • The enhanced sequence coverage facilitates a more comprehensive understanding of protein structure and modifications.