4.8 Article

Extending Native Top-Down Electron Capture Dissociation to MDa Immunoglobulin Complexes Provides Useful Sequence Tags Covering Their Critical Variable Complementarity-Determining Regions

期刊

ANALYTICAL CHEMISTRY
卷 93, 期 48, 页码 16068-16075

出版社

AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c03740

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资金

  1. Netherlands Organization for Scientific Research (NWO) TTW project [15575]
  2. NACTAR project [16442]
  3. Spinoza Award [SPI.2017.028]
  4. European Union's Horizon 2020 research and innovation program [686547]
  5. [ENPPS.LIFT.019.001]

向作者/读者索取更多资源

Native top-down mass spectrometry, utilizing electron capture dissociation, has been applied to analyze intact oligomeric immunoglobulins. Despite structural differences, the top-down electron capture dissociation spectra of these immunoglobulins are similar and provide informative sequence tags for identification. This approach shows potential for characterizing and identifying IgMs in plasma, which are crucial for the immune response to pathogens.
Native top-down mass spectrometry (MS) is gaining traction for the analysis and sequencing of intact proteins and protein assemblies, giving access to their mass and composition, as well as sequence information useful for identification. Herein, we extend and apply native top-down MS, using electron capture dissociation, to two submillion Da IgM- and IgG-based oligomeric immunoglobulins. Despite structural similarities, these two systems are quite different. The similar to 895 kDa noncovalent IgG hexamer consists of six IgG subunits hexamerizing in solution due to three specifically engineered mutations in the Fc region, whereas the similar to 935 kDa IgM oligomer results from the covalent assembly of one joining (J) chain and 5 IgM subunits into an asymmetric pentamer stabilized by interchain disulfide bridges. Notwithstanding their size, structural differences, and complexity, we observe that their top-down electron capture dissociation spectra are quite similar and straightforward to interpret, specifically providing informative sequence tags covering the highly variable CDR3s and FR4s of the Ig subunits they contain. Moreover, we show that the electron capture dissociation fragmentation spectra of immunoglobulin oligomers are essentially identical to those obtained for their respective monomers. Demonstrated for recombinantly produced systems, the approach described here opens up new prospects for the characterization and identification of IgMs circulating in plasma, which is important since IgMs play a critical role in the early immune response to pathogens such as viruses and bacteria.

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