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A new LC-NETD-MS/MS method enables detailed sequencing of heparan sulfate (HS) oligosaccharides. This technique accurately characterizes complex HS structures, including isomers and epimers, advancing biological studies.

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Area of Science:

  • Glycoscience
  • Analytical Chemistry
  • Biochemistry

Background:

  • Heparan sulfate (HS) is crucial for physiological processes, mediating protein interactions.
  • Current HS analysis methods like LC-MS struggle with heterogeneity and labile sulfate groups, limiting detailed sequencing.
  • Understanding HS structure is vital for both normal physiology and disease states.

Purpose of the Study:

  • To develop and validate an online LC-MS/MS method for sequencing heparan sulfate (HS) oligosaccharides.
  • To overcome challenges in analyzing heterogeneous HS modifications and labile sulfate groups.
  • To enable precise structural characterization of HS, including isomers and epimers.

Main Methods:

  • Utilized hydrophilic interaction liquid chromatography (HILIC) coupled with negative electron transfer dissociation (NETD) for online LC-MS/MS sequencing.
  • Employed an online cation exchange device (ion suppressor) to enhance precursor charge states.
  • Analyzed synthetic HS oligosaccharides with varying chain lengths, sulfation patterns, and uronic acid epimerization.

Main Results:

  • Successfully separated and sequenced HS oligosaccharides based on their fine structure and elution order.
  • NETD generated abundant glycosidic fragments, enabling characterization of both lowly and highly sulfated HS.
  • Diagnostic cross-ring ions differentiated specific sulfation patterns (6-O vs. 3-O), allowing unambiguous structural assignment.

Conclusions:

  • The developed LC-NETD-MS/MS method is a powerful tool for sequencing heterogeneous HS mixtures.
  • This technique can differentiate isomers, epimers, and characterize diverse sulfation motifs, including rare ones.
  • LC-NETD-MS/MS holds significant potential for advancing biological studies involving HS structure-function relationships.