Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin‐I for capturing sub‐glycoproteomics from breast cancer and disease‐free human sera

Abstract

In this study, a liquid-phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub-glycoproteomics that are affected in cancers, e.g. breast cancer. The platform is comprised of three monolithic columns with surface immobilised lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con A have specificities directed towards the core portion of N-glycans on the glycoprotein surface, RCA-I specifically interacts with the non-reducing terminal moieties of the outer chain structures of N-glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA → Con A → RCA-I (denoted as WCR) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease-free and breast cancer sera, respectively, corresponding to 75 and 65 non-redundant proteins, respectively. Using mass spectral count ratios and Q-Q plots yielded a panel of 23 non-redundant differentially expressed proteins (i.e. a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate.

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

“…The majority of the reported monolithic columns for separation and analysis of glycans are organic polymer-based [82][83][84][85][86][87]89,90,[92][93][94][95][97][98][99][100][101][102][103][104][107][108][109][110][111][112]114,115,[118][119][120][121]123,[144][145][146]. This may be due to the straightforward approach in the preparation and the availability of a variety of functional monomers to obtain desired functionality of the surface.…”

“…Fused silica capillaries with inner diameter that ranges from 75 µm to 530 µm were the most commonly used packing material in these monolithic columns [82-85,87-94,96,98,99,102-105, 107-110,114,115,117-120,123,144]. Other types such as stainless steel tube [95,111,121], poly(etherether-ketone) (PEEK) column [146], pipette tips [100,101] and syringe [106] have also been reported.…”

“…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.…”

“…The analysis was done by LC-MS/MS. Using this threedimensional set-up, 23 candidate biomarkers for breast cancer were obtained through MS spectral counts data [121]. In another work, a comprehensive separation using two-dimensional set-up consisted of RP and ZIC-HILIC monolithic columns were designed and integrated with ESI-MS and ESI-MS/MS analysis.…”

Recent Developments in Liquid Chromatography and Capillary Electrophoresis for the Analysis of Glycoprotein Glycans

“…69 Lectin affinity columns have been applied to determine cancer-related glycans in human plasma. 70,71 Lectin-immobilized nanoparticles have been used to entrap glycoproteins in human body fluids. 72 The capture of glycoprotein glycans using hydrazide functionalized phases was first introduced by Zhang's group.…”