Detection of Glycomics and Glycoproteomics using ZIC^®-HILIC

Analysis of protein glycosylation using EMD Millipore’s SeQuant^® ZIC^®-HILIC technology

SeQuant^® ZIC^®-HILIC sorbent is suitable for separation and extraction of glycans and glycopeptides. A material pore size of 200 Å is often appropriate 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 set-ups, whereas larger conventional column dimensions fit better to separations of labelled glycan structures detected with other techniques.

Below is an introduction to this field of analysis, including several suitable procedures and links to scientific papers.

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. Fractionation and enrichment of the glycosylated species are consequently 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).

Using EMD Millipore’s SeQuant^® ZIC^®-HILIC technology for the analysis of protein glycosylation

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 content 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 and 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 often-used 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.

Various HILIC applications for the analysis of released (free) glycans and glycopeptides have been reported ranging 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.

More detailed information is available in the Technical Summary and in the scientific papers listed below.

Suggested further reading:

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

Visit our on-line tutorial to learn more about HILIC technology or request our comprehensive „A Practical Guide to HILIC“ booklet. Both provide detailed explanations of technology and techniques to ensure your success using EMD Millipore’s innovative SeQuant^® ZIC^®-HILIC products.

If you still can’t find the information you need or want to publish any novel results of your own, you can always contact the SeQuant^® Support Team who are happy to assist.

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