Glycan node analysis

A bottom-up approach to glycomics

Sahba Zaare, Jesús S. Aguilar, Yueming Hu, Shadi Ferdosi, Chad Borges

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GCMS) can routinely detect the collection of branch-point and linkage-specific monosaccharides (“glycan nodes”) present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

Original languageEnglish (US)
Article numbere53961
JournalJournal of Visualized Experiments
Volume2016
Issue number111
DOIs
StatePublished - May 22 2016

Fingerprint

Glycomics
Sugar Alcohols
Polysaccharides
Glycosyltransferases
Hexoses
Acetates
Mass spectrometry
Enzymes
Acetylation
Gas Chromatography-Mass Spectrometry
Liquids
Gene expression
Gas chromatography
Condensation
Liquid-Liquid Extraction
Monosaccharides
Glycoside Hydrolases
Neoplasm Genes
Limit of Detection
Ions

Keywords

  • Cancer
  • Chemistry
  • Gas chromatography (GC)
  • Glycans
  • Glycobiology
  • Glycoproteins
  • Glycosyltransferase
  • Issue 111
  • Mass spectrometry (MS)
  • N-linked glycans
  • O-linked glycans
  • Permethylation

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

Glycan node analysis : A bottom-up approach to glycomics. / Zaare, Sahba; Aguilar, Jesús S.; Hu, Yueming; Ferdosi, Shadi; Borges, Chad.

In: Journal of Visualized Experiments, Vol. 2016, No. 111, e53961, 22.05.2016.

Research output: Contribution to journalArticle

Zaare, Sahba ; Aguilar, Jesús S. ; Hu, Yueming ; Ferdosi, Shadi ; Borges, Chad. / Glycan node analysis : A bottom-up approach to glycomics. In: Journal of Visualized Experiments. 2016 ; Vol. 2016, No. 111.
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