Automated Platform for Fractionation of Human Plasma Glycoproteome in Clinical Proteomics

Abstract

This publication describes the development of an automated platform for the study of the plasma glycoproteome. The method consists of targeted depletion in-line with glycoprotein fractionation. A key element of this platform is the enabling of high throughput sample processing in a manner that minimizes analytical bias in a clinical sample set. The system, named High Performance Multi Lectin Affinity Chromatography (HP-MLAC), is composed of a serial configuration of depletion columns containing anti-albumin antibody and Protein A with in-line multi lectin affinity chromatography (M-LAC) which consists of three mixtures of lectins Concanavalin A (Con A), Jacalin (JAC) and Wheat Germ Agglutinin (WGA). We have demonstrated that this platform gives high recoveries for the fractionation of the plasma proteome (≥95%) and excellent stability (over 200 runs). In addition, glycoproteomes isolated using the HP-MLAC platform were shown to be highly reproducible and glycan specific as demonstrated by re-chromatography of selected fractions and proteomic analysis of the unbound (glycoproteome 1) and bound (glycoproteome 2) fractions.

Post-translational Modifications and Mass Spectrometry Detection

“…Moreover, lectins can be immobilized in distinct solid supports (e.g. silica (157), agarose (158) or sepharose (159)), monolithic columns (160), and in microdimensional systems (146,153,156,161,162), including magnetic particles (142,153). These magnetic beads coupled to lectins have been recently used for capturing glycoproteins in complex biological samples, taking advantage of their high surface area and high mobility in solution (142,161).…”

Targeted High-Throughput Glycoproteomics for Glyco-Biomarker Discovery

“…M-LAC was coupled with 1D SDS-PAGE, isoelectric focusing and lectin-overlay antibody microarray to identify several glycoproteins such as alpha-1B-glycoprotein and complement C3 as potential candidates (Zeng et al 2011). Kullolli et al further developed M-LAC into a high performance multi-lectin affinity chromatography (HP-MLAC), involving targeted albumin and immunoglobulin depletion in-line with glycoprotein affinity isolation using M-LAC (Kullolli, Hancock, and Hincapie 2010). This method has shown reproducibility and consistency of the bound and unbound fraction over 200 runs which promises to provide quality plasma glycoproteome data for clinical proteomics.…”

Flow Lectin Affinity Chromatography−A Model with Sambucus nigra Agglutinin

“…An advantage over antibodies is that lectins detect glycan structures in glycoproteins, which is useful since many disease biomarkers are glycoproteins with aberrant glycans, namely in cancer [7][8][9]. Another benefit compared to antibodies is that the affinity of lectins to glycans is lower than the corresponding antibody-antigen interactions, so elution of bound glycoproteins is more efficient and higher recovery rates are achieved with lectins [10]. Other advantages over antibodies include the cost of the enrichment column, the life-time and the loading capacity of lectin columns versus antibody columns [10].…”

“…Another benefit compared to antibodies is that the affinity of lectins to glycans is lower than the corresponding antibody-antigen interactions, so elution of bound glycoproteins is more efficient and higher recovery rates are achieved with lectins [10]. Other advantages over antibodies include the cost of the enrichment column, the life-time and the loading capacity of lectin columns versus antibody columns [10].…”

“…Although there is a rising tendency to automate analytical procedures used in proteomic research through flow methodologies [10,[16][17][18][19], usually LAC is carried out in batch conditions (using the commercial columns and kits) [20] or using sophisticated chromatographic equipments [21][22][23], which implies a long procedure time and high consumption of consumables, or capability of laboratories to purchase expensive apparatus. If the LAC procedure is performed under flow conditions, this allows automation of the process, especially important when several samples need to be run, and reduces solution and sample manipulation steps and resultant losses, ex-vivo proteolysis or chemical modifications during procedures [10].…”