Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow

Anal Chem. 2016 May 3;88(9):4795-802. doi: 10.1021/acs.analchem.6b00259. Epub 2016 Apr 13.


The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery.

MeSH terms

  • Animals
  • Cells, Cultured
  • Chromatography, High Pressure Liquid
  • Glycoside Hydrolases / metabolism*
  • Glycosphingolipids / analysis*
  • Glycosphingolipids / metabolism
  • HeLa Cells
  • High-Throughput Screening Assays*
  • Humans
  • Mice
  • NIH 3T3 Cells
  • Polysaccharides / analysis*
  • Polysaccharides / metabolism
  • Rhodococcus / enzymology
  • Substrate Specificity


  • Glycosphingolipids
  • Polysaccharides
  • Glycoside Hydrolases
  • endoglycoceramidase