Developing High Resolution HILIC Separations of Intact Glycosylated Proteins Using a Wide-Pore Amide-Bonded Stationary Phase

Library Number:
APNT134848058
Part Number:
720005380EN
Author(s):
Matthew A. Lauber, Scott A. McCall, Bonnie A. Alden, Pamela C. Iraneta, and Stephan M. Koza
Source:
Waters
Content Type:
Application Notes
Content Subtype:
Application Notes
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Hydrophilic interaction chromatography (HILIC) has been widely adopted as a tool for separating highly polar compounds, and it has become a common technique for small molecule separations. In contrast, use of HILIC for large biomolecules has been comparatively limited even though there are instances in which the selectivity of HILIC would be highly valuable, for example, during the characterization of protein glycosylation.

A typical approach to the analysis of glycans involves their enzymatic or chemical release from the protein followed by chromatographic separation by HILIC. UPLC-based separations, founded on an optimized sub-2-µm amide-bonded stationary phase has transformed HILIC separations of released glycans by facilitating faster, high-resolution separations.

To enable the analysis of glycans that are still attached to the protein, we present in this application note an optimized HILIC column and corresponding methods for resolving the glycoforms of intact and digested glycoproteins ranging in mass from 10 to 150 kDa. A wide-pore (300Å) amide-bonded, organosilica (ethylene bridged hybrid, or BEH) stationary phase is employed.

Just as reversed-phase separations are employed for resolving protein isoforms that have varying hydrophobicities, we demonstrate that HILIC separations with BEH Amide 300Å can be used for resolving protein isoforms that exhibit varying hydrophilicities, such as isoforms differing with respect to glycan occupancy.

With the availability of these new separation capabilities, it will be possible to perform more detailed characterization of intact glycoproteins, whether by means of combining HILIC with optical detection or with ESI-MS.


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