Automating Middle-Down Mass Spectrometry Analysis for Extensive Antibody Characterization.

Area of Science:

• Proteomics
• Biopharmaceutical Analysis
• Mass Spectrometry

Background:

• Peptide mapping is widely used but lacks proteoform information.
• Top-down and middle-down workflows offer deeper proteoform insights but are complex.
• Existing software often requires multiple tools for targeted protein characterization.

Purpose of the Study:

• To introduce Proteoform Studio, an automated platform for routine intact protein characterization.
• To streamline top-down and middle-down mass spectrometry workflows.
• To reduce user expertise and data analysis bottlenecks in proteoform analysis.

Main Methods:

• Development of Proteoform Studio, an integrated software platform.
• Automated data processing including instrument run setup, method modification, data deconvolution, and proteoform searching.
• Automated aggregation of fragmentation results for enhanced characterization.
• Optimization of fragment ion matching parameters (signal-to-noise ratio, isotope fit score).

Main Results:

• >80% sequence coverage of antibody light chain (Lc) and N-terminal heavy chain (Fd) subunits achieved via automated middle-down analysis.
• Automated matching results aligned well with manual validation, reducing manual verification needs.
• Aggregation of spectral results from complementary fragmentation methods improved sequence coverage.

Conclusions:

• Proteoform Studio enables routine, automated intact protein characterization with a single software tool.
• The software lowers barriers to entry and eliminates data analysis bottlenecks for biopharmaceutical assays.
• Automated analysis drives adoption and enables more precise characterization of therapeutic proteoform landscapes.