Monolithic silica capillary columns having immobilized lectins and surface bound polar functionalities for lectin affinity and normal phase nano‐LC and CEC of glycoconjugates, respectively

Abstract

In this report, monolithic silica-based capillary columns were produced by the sol-gel process and subsequently silanized with gamma-glycidoxypropyltrimethoxysilane to form on the surface of the monolith a reactive gamma-glycidoxypropylsilyl sublayer to which an interactive top layer can be covalently attached. The interactive top layer consisted of either an immobilized lectin or polar cyano functions to perform lectin affinity chromatography (LAC) of glycoproteins or normal phase chromatography (NPC) of glycans, respectively. Two lectins were immobilized, namely Con A and wheat germ agglutinin (WGA) due to their utility in LAC of a wide range of glycoconjugates. On the other hand, 1H-imidazole-4,5-dicarbonitrile was covalently attached to the silica monolith to yield polar silica monolith referred to as 2CN-OH monolith. The two lectin monolithic columns were evaluated in Nano-LC with standard glycoproteins over a wide range of conditions and proved useful in capturing the glycoforms bearing the pertinent saccharidic motifs for interaction with the given lectin. The 2CN-OH monolithic columns were effective in the profiling of glycans derived from glycoproteins in both NP-CEC and NP-Nano-LC with NP-CEC offering enhanced separation when compared to NP-Nano-LC.

Development of Monolithic Column Materials for the Separation and Analysis of Glycans

“…An example of post-modification was photografting of hydrophilic polymer poly(ethyleneglycol) methacrylate (PEGMA) to reduce the hydrophobicity of the monolith [82]. To create affinity mode, lectins were immobilized on the surface in various ways such as covalent binding of amine residues to aldehydefunctionalized surfaces [84,98,115,117,[119][120][121], chelation to (IDA-Cu 2+ ) modified surfaces [99] and a molecular imprinting method using a polydopamine coating [112]. Coating a monolith surface with nanoparticles provides a high loading of functional groups, fixing the problem of limited active sites described above.…”

“…It was utilized to immobilize lectins (Con A and WGA) to provide a lectin affinity mode for LC separation of glycoproteins and also modification with 1H-imidazole-4,5-dicarbonitrile to provide polar cyano-mode for CEC separation. Both modes of separation were optimized and tested for their respective separation functions [117].…”

Monolithic media for applications in affinity chromatography

“…Lectin AC relies on the specificity of sugar‐binding proteins toward defined carbohydrate residues (see above) 99–101. Silica monoliths with immobilized Con A and wheat germ agglutinin (WGA) were successfully applied for separating glycoproteins based on their glycosylation patterns 102. A methacrylate monolith modified with iminodiacetic acid (IDA) for the Cu 2+ ‐mediated immobilization of Con A was used for the enrichment of glycoproteins from diluted urine 103.…”

Macroporous monolithic supports for affinity chromatography

“…Finally, to prove the application of these affinity columns in the profiling of low amounts of complex sample, these capillary columns were successfully implemented in the elucidation of glycoprotein from mouse urine samples. Zhong and El Rassi reported the immobilization of Con A and WGA on silica‐based monolithic supports 60. The silica monolith was prepared by the sol‐gel process, and γ‐glycidoxypropyltrimethoxysilane was further grafted to obtain epoxy groups in the monolithic surface.…”