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Toward a platform for comprehensive glycan sequencing.

Vernon Reinhold1, Hailong Zhang, Andrew Hanneman

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A new analytical platform offers quantitative and qualitative N- and O-glycosylation analysis, moving beyond basic understanding to detailed structural insights for improved glycomics research.

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Area of Science:

  • Glycomics
  • Analytical Chemistry
  • Biochemistry

Background:

  • Glycosylation is crucial for protein function and disease.
  • Current methods lack comprehensive structural detail and quantitative accuracy.
  • A need exists for integrated platforms for detailed glycan analysis.

Purpose of the Study:

  • To present an integrated analytical platform for quantitative and qualitative N- and O-glycosylation analysis.
  • To enable detailed structural understanding, including peptide-glycan connectivity.
  • To establish a best-methods approach for advancing glycomics.

Main Methods:

  • Sequential enzymatic N-glycan and chemical O-glycan release from glycopeptides.
  • Solid-phase extraction and derivatization with a pyrazolone analog for labeling.
  • Methylation and mass spectrometry (MS(n)) for structural elucidation and quantification.

Main Results:

  • Developed a platform for comparative quantification of glycosylation.
  • Demonstrated stability of glycans to methylation for sequential disassembly.
  • Applied the platform to human serum and cancer samples, characterizing specific epitopes like sLe(x).

Conclusions:

  • The integrated platform provides comprehensive structural detail of N- and O-glycosylation.
  • Enables collaborative advancement of glycomics protocols.
  • Confirms structural details using synthetic standards and characterized biologics via MS(n).