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Rapid high-throughput antibody analysis using microwave-assisted digestion.

Thais Mingatos de Toledo1, Hellen Paula Valerio1, Antônio Moreira Marques Neto2

  • 1Laboratory of Glycoproteomics, Parasitology Department, Biomedical Science Institute, São Paulo University, São Paulo, Brazil.

Journal of Proteomics
|January 22, 2026
PubMed
Summary
This summary is machine-generated.

Microwave-assisted digestion combined with mass spectrometry offers a rapid, reliable method for antibody analysis, significantly reducing deamidation and improving peptide identification for comprehensive characterization.

Keywords:
AntibodiesData-dependent acquisitionData-independent acquisitionMicrowave-assisted digestionPost-translational modifications

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Area of Science:

  • Biochemistry
  • Analytical Chemistry
  • Protein Science

Background:

  • Monoclonal antibodies are crucial biotherapeutics requiring robust quality control.
  • Multi-attribute methods (MAM) using mass spectrometry are vital for assessing critical quality attributes (CQAs).
  • Traditional protein digestion methods can be time-consuming and introduce artifacts.

Purpose of the Study:

  • To develop and optimize a rapid microwave-assisted (MW) digestion workflow for antibody samples.
  • To evaluate the impact of different buffers and pH on MW digestion efficiency and artifact formation.
  • To compare data-dependent acquisition (DDA) and data-independent acquisition (DIA) for comprehensive antibody characterization.

Main Methods:

  • Development of a high-throughput (96-sample) mass spectrometry workflow.
  • Evaluation of microwave-assisted protein digestion under various buffer conditions and pH.
  • Analysis using both DDA and DIA, coupled with peptide and post-translational modification (PTM) probing.

Main Results:

  • MW-assisted digestion provided fast, reliable protein digestion with high sequence coverage and minimized artifacts.
  • DIA combined with MW digestion enhanced peptide identification and PTM detection compared to DDA.
  • Sodium acetate buffer under MW conditions was most effective in reducing deamidation and oxidation.

Conclusions:

  • MW-assisted digestion is a superior alternative to conventional methods for antibody sample preparation.
  • The optimized MW digestion workflow combined with DIA enables comprehensive and unbiased antibody characterization.
  • Further optimization is needed to address protein oxidation, but the method significantly reduces other artifacts like deamidation.