Abstract
Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC)-mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time) are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS) analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin) employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization.
Highlights
Proteins are macromolecules with a complex and heterogeneous structure, which is partly due to post-translationalଝ Selected papers from the 32nd International Symposium on Chromatography (ISC 2018), September 23–27, 2018, Cannes-Mandelieu, France.G. van Schaick et al / J
The hydrophilic-interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC) columns had the same dimensions and particle characteristics, and the same solvents A and B were used for both separation approaches
Protein samples were prepared in water and analyzed using linear gradients from 20% B to 50% B in 30 min and from 95% B to 40% B in 30 min when using HILIC and RPLC, respectively
Summary
Proteins are macromolecules with a complex and heterogeneous structure, which is partly due to post-translational. An attractive alternative for the separation and characterization of intact glycoproteins is hydrophilic-interaction liquid chromatography (HILIC) [14] This technique is mostly applied to the chromatographic separation of small polar molecules [15] peptides and glycopeptides [16]. To facilitate efficient method development in LC, computer-aided approaches, such as ChromSword, DryLab and PIOTR, have been developed [22,23,24] These software use models (based on, e.g., linear solvent strength (LSS), ion exchange or mixed-mode) to describe retention and to make predictions of protein retention times based on a limited number of scouting gradients. A PIOTR method employing the HILIC adsorption retention model and a Paretooptimization approach was evaluated as a prediction tool to obtain gradient separation conditions of glycoforms from proteins varying in degree and complexity of glycosylation (Fc parts of trastuzumab, ovalbumin, fetuin, alpha-1-acid glycoprotein, and thyroglobulin). Gradient conditions resulting in efficient glycoform separations could be readily derived from scouting gradients, which by itself did not provide glycoform resolution
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