Flow Lectin Affinity Chromatography−A Model with Sambucus nigra Agglutinin

Abstract

The study of proteomes has become an important way to assess changes occurring in disease conditions. Nonetheless, the dominance of highly abundant serum proteins significantly complicates the discovery and analysis of low-abundant glycoproteins that may constitute potential disease biomarkers. Several techniques have been used to perform serum proteome partitioning such as affinity chromatography based on protein A and G, avian IgY, monoclonal antibodies and lectins. Lectins are particularly well-suited to bind and selectively isolate certain glycan structures present in complex samples like biological ones. The lectin reversibly captures glycoproteins with a particular glycostructure present in the sample in different abundance rates, yielding, after elution, an enriched pool of glycoproteins for glycoproteomic studies. However, lectin affinity chromatography (LAC) for biochemical applications is mainly carried out in batch conditions, which implies a long procedure time, expensive reagents or columns and special equipments or installations. This work proposes the development of a lab-made and easy to implement flow LAC procedure, using Sambucus nigra agglutinin (SNA) immobilized on agarose beads to isolate the STn glycan (a pan-carcinoma biomarker) present in glycoproteins of cancer patients serum. Under flow conditions, the proposed LAC procedure allows the automation of the process, especially important when several samples need to be run. Additionally, the flow system does not require expensive installations or experienced personnel, and it can be tailored to perform LAC with any lectin, according to the purpose of the glycoproteomic analysis. Significance: This work aims to demonstrate the feasibility and the benefits of performing lectin affinity chromatography under flow conditions and with a very simple flow manifold, when compared with the batch procedure usually carried out in Biochemistry and Molecular Biology laboratories. The easiness to implement this simple manifold, without requiring expensive equipments or installations, allows its use in any laboratory, even in those with financial limitations. Several advantages of the developed methodology are pointed out, versus the use of batch procedures or commercial kits or columns, especially when routine analyses need to be performed.