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Comprehensive Proteoform Characterization of Plasma Complement Component C8αβγ by Hybrid Mass Spectrometry Approaches

Vojtech Franc1,2, Jing Zhu1,2, Albert J R Heck3,4

  • 1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Padualaan 8, 3584 CH, Utrecht, The Netherlands.

Journal of the American Society for Mass Spectrometry
|March 14, 2018
PubMed

Insights

The human complement C8 protein

Area of Science:

  • Immunology and protein biochemistry.

Background:

  • The complement C8 protein complex is crucial for membrane attack complex (MAC) formation and function.
  • Understanding C8's structural heterogeneity is key to elucidating its role in complement-mediated lysis.

Purpose of the Study:

  • To comprehensively characterize the structural micro-heterogeneity and post-translational modifications of human C8 protein.
  • To identify novel glycosylation sites and PTMs on C8 subunits.

Main Methods:

  • High-resolution native mass spectrometry (MS) and peptide-centric proteomics.
  • Ion exchange chromatography for C8 fractionation.
  • Liquid chromatography-tandem mass spectrometry (LC-MS/MS) for site-specific glycan analysis.

Main Results:

  • Identified at least 20 co-occurring C8 proteoforms using native MS.
  • Discovered two novel N-glycosylation sites on C8.
  • Provided the first experimental evidence of O-linked glycans on the C8γ subunit.
  • Elucidated the stoichiometry of C-mannosylation sites on C8α and C8β subunits.

Conclusions:

  • Detailed structural micro-heterogeneity and diverse post-translational modifications (PTMs) of human C8 were characterized.
  • Newly identified PTMs, particularly O-glycosylation on C8γ, may influence MAC formation and function.
  • This provides a comprehensive proteoform specification of C8 with implications for complement biology.
Keywords:
C-glycosylationComplement component C8Glycopeptide-centric proteomicsGlycosylationIon-exchange chromatographyMembrane attack complexN-glycosylationNative mass spectrometryO-glycosylationPlasma proteins

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