Detection of Glycomics and Glyco- proteomics using SeQuant® ZIC®-HILIC


Simple Identification of Carbohydrates

SeQuant® ZIC® is the ideal sorbent for the separation and extraction of glycans and glycopeptides. A material pore size of 200 Å is recommended to avoid size-exclusion effects from the relatively large hydrodynamic volume of the hydrated glycan structures. Small ID HPLC columns are suitable for LC-MS methods, whereas larger conventional column dimensions are more appropriate for separations of labeled glycan structures detected with other techniques.

Introduction to Protein Glycomics and Glycoproteomics

The attachment of monosaccharides to proteins, known as protein glycosylation, is an abundant post-translational modification (PTM). It is widely accepted that protein glycosylation is involved in numerous essential cellular processes. Thus, the structural and functional characterizations of these PTMs are important in the rapidly growing research areas of glycomics and glycoproteomics.

One of the most prominent challenges associated with these disciplines is the substoichiometric presence of the glycosylation, resulting from substantial heterogeneity of the attached carbohydrates (collectively called glycans) as well as only partial glycan occupancy of a given site. Consequently, fractionation and enrichment of the glycosylated species are essential to alleviate this problem. However, the significant hydrophilicities associated with these biomolecules, which arise from the numerous hydroxyl groups of the glycan, limit the use of traditional chromatographic techniques such as reversed-phase liquid chromatography (RPLC).

Advantages of HILIC Technology

In contrast, HILIC (hydrophilic interaction liquid chromatography) is an attractive chromatographic technique for the analysis of these glycoconjugates. Applications usually involve detection by mass spectrometry (MS) to benefit from its high sensitivity, accuracy and resolution as well as its high throughput potential. Several HILIC stationary phases with different functional groups are available, and solvents with a high concentration of organic component (40-97%) in water are typically used for the mobile phase. Since it can be difficult to dissolve glycans and glycopeptides if the amount of organic component is very high, 80% organic content is often used in the mobile phase as a starting condition.

Acetonitrile is by far the most popular organic modifier. To hydrate the stationary phase and generate reproducible results, at least 3% water should be present. Suitable buffers include ammonium salts of acetate and formate due to their volatibility and excellent solubility in organic solvent. These salts have been shown to minimize the electrostatic interactions (repulsions or attractions) between charged stationary phases and analytes i.e. sialic acids containing glycoconjugates. Formate and other weak acids are common mobile phase additives for adjusting pH when using on-line HILIC-MS setups. An overview of the typical workflows in glycomics and glycoproteomics is shown in the figure below.

Typical Workflows in Glycoproteomics and Glycomics

Steps involving HILIC are highlighted in red.

Merck:/Freestyle/LE-Lab-Essentials/Chromatography/LE-workflows in glycoproteomics and glycomics-201007.181.jpg
Columns for Glycans & Glycopeptides
Further Reading

SeQuant® ZIC®-HILIC HPLC Technology 
(3,5µm, 200 Å) 150×0,3 mm
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SeQuant® ZIC®-HILIC HPLC Technology
(3,5µm, 200 Å) 150×1,0 mm
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SeQuant® ZIC®-HILIC HPLC Technology
(3,5µm, 200 Å) 150×2,1 mm
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SeQuant® ZIC®-HILIC HPLC Technology
(5µm, 200 Å) 150×4,6 mm
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Various HILIC applications for the analysis of released (free) glycans and glycopeptides have been contributed. These range from sample preparations using HILIC in solid-phase extraction (SPE) formats to chromatographic separation using analytical and capillary-scale HILIC columns with both off- and on-line MS detection.
Detailed information is available in the scientific papers listed below.

SeQuant® ZIC®-HILIC HPLC Technology HPLC Technology
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Analysis of Protein Glycosylation using HILIC
Thaysen-Andersen M.
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Analysis of Protein Glycosylation and Phosphorylation using HILIC-MS
Thaysen-Andersen M, Engholm-Keller K, Roepstorff P., Ed: Wang P and He W, Taylor and Francis. 2011
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Structural glycomics using hydrophilic interaction chromatography (HILIC) with mass spectrometry
Wuhrer M, de Boer A, Deelder AM, Mass Spectrom. Rev. 28, 192-206, 2009
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