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Extending Serum IgG1 Antibody Repertoire Coverage Using DIA-PTCR
Rien W Leuvenink1, Linus Wollenweber1, Albert Bondt1
1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8 3584 CH, Utrecht, The Netherlands.
Gas-phase fractionation using data-independent analysis with proton transfer charge reduction (DIA-PTCR) significantly enhances antibody repertoire profiling. This method doubles the number of detectable antibody clones in serum by reducing spectral congestion in LC-MS analysis.
Area of Science:
- Biochemistry
- Immunology
- Analytical Chemistry
Background:
- Liquid chromatography-mass spectrometry (LC-MS) based antibody Fab profiling is crucial for understanding immune responses.
- Extreme spectral congestion in LC-MS hinders the detection and quantification of diverse antibody clones in serum.
- Current methods face challenges in comprehensively analyzing antibody repertoires due to molecular similarities and ionization complexities.
Purpose of the Study:
- To develop a method for reducing spectral congestion in LC-MS-based Fab profiling.
- To enhance the number of detectable antibody clones within a single LC-MS run.
- To maintain quantitative accuracy for antibody abundance measurements in repertoire profiling.
Main Methods:
- Implementation of gas-phase fractionation using proton transfer charge reduction (PTCR) in a data-independent analysis (DIA) acquisition mode (DIA-PTCR).
- Utilizing a limited number of isolation windows (five) within a specific m/z region to cover the total Fab distribution.
- Integration of reduced noise filtering alongside gas-phase fractionation.
Main Results:
- The DIA-PTCR method significantly reduced spectral congestion, a major challenge in LC-MS Fab profiling.
- The number of detected antibody clones per run increased by over 100%, from 1043 to 2063.
- Quantitative data on antibody abundances were successfully retained, enabling accurate repertoire profiling.
Conclusions:
- DIA-PTCR is a highly beneficial technique for advancing LC-MS-based antibody repertoire profiling.
- This approach overcomes limitations of spectral congestion, enabling deeper insights into antibody diversity.
- The method offers a more comprehensive and quantitative analysis of antibody repertoires.
