Complex Carbohydrate Research Center - Analytical Services

• Glycosyl composition
• Glycosyl linkage
• Molecular weight
• Glycan profiling
• 1D and 2D-NMR
• Mass spectrometry (MALDI-MS, ESI/NSI-MS and MS/MS, LC-ESI-MS, GC-MS)

We can perform glycomics studies starting from either purified glycoproteins or from cells, tissues, culture supernatants, and gel slices. The analysis can begin on purified animal, plant or bacterial, natural or recombinant glycoproteins, or we can isolate and purify the glycoprotein mixture from cells, tissues, culture supernatants, or gel slices. We can develop custom glycomics services as needed.

We offer full structural characterization of glycosaminoglycan products and biosimilars such as heparin, low molecular weight heparin, chondroitin sulfate and other GAGs.

Services being offered for GAG structural analysis:
• Enzyme digestion of glycosaminoglycans to disaccharides
• Chemical degradation and HPLC, MS or NMR analysis
• Oligosaccharide profiling by CTA-SAX-HPLC
• Identification of disaccharides and sulfation pattern by online ESI-MS and ESI-MS/MS
• Identification of released disaccharides by SAX-HPLC, and detection of the disaccharides by both UV and fluorescent detection.
• Structural characterization of GAG oligosaccharides by 1D and 2D NMR
• Sequencing of GAG oligosaccharides by ESI-MS and ESI-MS/MS
• Glycosyl composition analysis of GAGs by GC-MS

Including starch, arabinoxylans, cellulose, chitin and pectins.

• Purification of plant polysaccharides and isolation of pectin cell wall
• Glycosyl residue composition analysis
• Identification of lipid anchors
• MW determination by size exclusion chromatography with light scattering detector (SEC-ELSD)
• Chitin analysis
• Sequencing of polysaccharides by 1D and 2D-NMR analysis
• Specific test for beta-1,3-glucan analysis
• Absolute configuration (D or L) of monosaccharides
• Fatty acid analysis by GC-MS

We offer a combination of the following chemical and instrumental analysis for profiling lignocellulosic biomass and pretreated biomass:

• Pyrolysis Molecular Beam Mass Spectroscopy (py-MBMS): analysis of lignin and C5 and C6 glycosyl compositions in the plant cell wall. Total lignin content, p-hydroxyphenyl (H), syringyl to guaiacol (S/G/H) ratios can be estimated directly from the spectra.
• Pyrolysis Gas Chromatography Mass Spectroscopy (py-GCMS): analysis/characterization of lignin structure in plant cell walls.

Isolation methods:

• for smooth LPS and LOS: hot phenol/water, detergent, EDTA-TEA extraction
• for rough LPS mutants: phenol-chloroform-petroleum ether method (PCP)
  Extraction methods may need modifications based on the specific nature of the project.

Purification methods:

• Ultracentrifugation
• Enzymatic digestion (nucleases, protease)
• De-lipidation
• Size exclusion chromatography (SEC) in associative and dissociative conditions
  The progress of purification is verified by SDS-/DOC-PAGE.

Analysis of O-chain and core region released from LPS:
Purification and size determination by FPLC SEC, high-performance anion exchange chromatography (HPAEC) and reverse phase chromatography; monosaccharide composition by GC-MS and GC-FID, GC-CI; glycosyl linkage of neutral and acidic sugars by method of partially methylated alditol acetates (PMAA); glycosyl linkage of of Kdo (2-keto-3-deoxy-octulosonic acid); determination of certain hexosaminuronic acids, deoxyheptulosaric acids, and pseudaminic acid; elucidation of glycosyl sequence and structure by 1D and 2D homo- and heteronuclear NMR, and high resolution MALDI-TOF MS and MS/MS, electrospray ionization (ESI-MS) mass spectrometry.

Isolation and purification of capsular polysaccharides from Gram-negative and Gram-positive bacterial cell; monosaccharide and fatty acid composition by GC-MS and GC-FID, GC-CI; glycosyl linkage analysis by method of partially methylated alditol acetates (PMAA); purification and size determination by FPLC size exclusion chromatography (SEC), high-performance anion exchange chromatography (HPAEC); structure of polysaccharide and diacylglycerol lipid anchor moieties by MALDI-TOF MS and MS/MS, electrospray ionization (ESI-MS) and by 1D and 2D homo- and heteronuclear NMR.

EPSs are isolated form bacterial culture media or from biofilm either in your laboratory or in our facility (please consult specific condition) and extensively purified by using size exclusion chromatography (SEC). EPS can be characterized by molecular size, monosaccharide composition, glycosyl linkage analysis by method of partially methylated alditol acetates (PMAA) and by 1D and 2D homo- and heteronuclear NMR; Comprehensive structural characterization often requires enzymatic depolymerization of EPS.

We offer routine 1D-1H NMR and 13C-NMR spectroscopy as well as 2D-NMR experiments such as COSY, TOCSY, HSQC.

Nuclear Magnetic Resonance (NMR) instruments:

• 300 MHz (2 channel, gradients)
• 500 MHz (89mm wide-bore magnet for solids and liquids, 3 channel, gradients)
• 600 MHz (3 channel, gradient)
• 800 MHz (3 channel, gradients)