Preparation of boronate-functionalized molecularly imprinted monolithic column with polydopamine coating for glycoprotein recognition and enrichment

Highlights

• A facile approach was developed for preparation of glycoprotein-imprinted monolithic column.

• The as-prepared imprinted monolith showed high affinity toward template glycoprotein.

• It was successfully applied to the selective enrichment of HRP from human serum sample.

Abstract

A novel imprinting strategy using reversible covalent complexation of glycoprotein was described for creating glycoprotein-specific recognition cavities on boronate-functionalized monolithic column. Based on it, a molecularly imprinted monolithic column was prepared by self-polymerization of dopamine (DA) on the surface of 4-vinylphenylboronic acid (VPBA)-based polymeric skeletons after reversible immobilization of horseradish peroxidase (HRP). Due to the combination of boronate affinity and surface imprinting of DA, the stable and accessible recognition sites in the as-prepared imprinted monolith could be obtained after the removal of the template, which facilitated the rebinding of the template and provided good reproducibility and lifetime of use. The recognition behaviors of proteins on the bare VPBA-based, HRP-imprinted and nonimprinted monolithic columns were evaluated in detail and the results showed that the HRP-imprinted monolith exhibited higher recognition ability toward the template than another two monolithic columns. Not only nonglycoproteins but also glycoproteins can be well separated with the HRP-imprinted monolith. In addition, the feasibility of the HRP-imprinted monolith, adopted as an in-tube solid phase microextraction (in-tube SPME), was further assessed by selective extraction and enrichment of HRP from human serum. The good results demonstrated its potential in glycoproteome analysis.

Introduction

Protein glycosylation, as one of the most common post-translational modifications, plays a vital role in many biological processes [1]. Numerous studies have revealed that many clinical biomarkers are glycoproteins. Mass spectrometry has proven to be a powerful tool in glycoproteomic research. However, it is almost impossible to directly determine the low abundant glycoproteins without any enrichment process. Great efforts to overcome this limitation have focused on hydrophilic interaction chromatography [2], [3], hydrazine chemistry [4], lectin-based affinity chromatography [5] or boronate affinity chromatography [6], [7], [8]. Although these methods have achieved some success, additional affinity approaches for the enrichment of glycoproteins are highly desirable.

Molecular imprinting is an affinity method for the synthesis of tailor-made recognition materials by copolymerizing suitable functional monomers in the presence of template. As a promising technique, it has attracted great interest, and has been widely applied in the field of separation, catalysis and sensors [9], [10], [11], [12]. However, to date, most of the successes have focused on small molecules, whereas the imprinting of proteins has been far less successful. There are many inherent problems with the protein hindering the advancement, including the huge molecular size, poor mass transfer, flexible conformation and solubility [13]. Although several strategies, such as metal-chelating imprinting [14], [15], epitope imprinting [16], [17], and surface imprinting [18], [19], [20], [21], have been proposed to solve these issues, the imprinting of proteins still remains a great challenge.

4-Vinylphenylboronic acid (VPBA), as one of boronic acid derivates, can readily form cyclic boronate complexes with vicinal diols under basic conditions and reversibly dissociated under acidic conditions. As such easy on/off reactivity favors the imprinting and removal of glycoprotein templates, boronic acids can be promising functional monomers for establishing a general approach for glycoprotein imprinting. Although boronic acids have been demonstrated in the past few years as functional monomers for protein imprinting [22], [23], [24], [25], most attempts [22], [23], [24] would prefer to use m-aminophenylboronic acid (APBA) as polymerizable reagent rather than affinity reagent. For example, Bossi et al. [22] was the first to publish the paper on surface imprinting of proteins (including glycoproteins and nonglycoproteins) by oxidative self-polymerization of APBA on the surface of polystyrene microplates. Besides, Bonini et al. [23] and Rick and Chou [24] also developed a series of poly(APBA)-coated imprinted polymers over a wide variety of supporting matrices for protein recognition based on the same principle. Until recently Liu's group [25] has developed a general and facile method for glycoprotein imprinting based on the principle of boronate affinity interaction, in which VPBA was used as the sole functional monomer to form a cyclic ester with a sugar moiety of glycoproteins. The resultant imprinted polymers showed preferential binding of the template glycoprotein over the competitive glycoproteins. Importantly, the formation of reversible covalent complex can provide good access to the imprinted sites, thus enable the elution and rebinding of the target glycoprotein easily. Nevertheless, the advantages of boronic acid-functionalized imprinted materials have not been full explored so far.

Monolithic column features several significant advantages compared to microparticles-packed column: low back pressure, fast mass transfer kinetics, high loading capacity and ease of preparation. The introduction of monolithic column to protein imprinting may be a breakthrough in developing new protein-imprinted materials without the tedious procedures such as grinding, sieving and packing that were often encountered in the preparation of packed columns. Although recent successful studies [26], [27], [28], [29] have demonstrated its feasibility for preparation of protein-imprinted monolithic columns since Hjertén et al. [30] first reported on in situ polymerization of imprinted monolithic column for protein recognition, no papers about molecularly imprinted monolithic columns for glycoprotein recognition are found so far. On the other hand, combination of boronate affinity with monolithic columns format can offer more functions for the analysis of glycoproteins. Although Liu's group [31], [32] and our group [6], [33] have recently developed a series of boronate affinity monolithic columns for glycoprotein recognition, making a clear distinction between glycoproteins is still rather difficult using the types of boronate affinity monoliths.

Herein, a new molecularly imprinted monolithic column was prepared by self-polymerization of dopamine (DA) on the surface of VPBA-based polymeric skeletons after reversible immobilization of the template horseradish peroxidase (HRP). The resultant imprinted monolithic column exhibited specific recognition toward HRP. Significantly, the imprinted monolith could separate HRP not only from nonglycoproteins, but also from other glycoproteins. In addition, the practicability of the imprinted monolithic column was demonstrated by using it as an in-tube solid phase microextraction (in-tube SPME) for selective enrichment of HRP from human serum sample.