Efficacy of glycoprotein enrichment by microscale lectin affinity chromatography

Abstract

Reproducible and efficient affinity enrichment is increasingly viewed as an essential step in many investigations leading to the discovery of new biomarkers. In this work, we have evaluated the repeatability of lectin enrichment of glycoproteins from human blood serum through both qualitative and quantitative proteomic approaches. In a comprehensive evaluation of lectin binding, we have performed 30 separate microscale lectin affinity chromatography experiments, followed by a conventional sample purification, and LC-MS/MS analysis of the enriched glycoproteins. Two lectin affinity matrixes, both with Con A lectin immobilized to the same solid support but differing in the amount of immobilized lectin, were investigated to characterize their binding properties. Both qualitative and quantitative data indicate acceptable repeatability and binding efficiency for the lectin materials received from two different commercial sources.

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).…”

“…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). Immobilization or covalent capture of the target molecules seems promising, since it allows extensive washing without significant loss in sensitivity (161). Taken together, when the aim is the broad range analysis of protein glycosylation, lectin arrays seem to be the most suitable strategy to cover different types of glycan structures and to study their relationship with cellular dynamics (143,163).…”

A quantitative investigation of fucosylated serum glycoproteins with application to esophageal adenocarcinoma

“…While both of these techniques provide a general method for glycoprotein enrichment, only the third strategy, namely lectin affinity, provides the potential to target subsets of glycans that may be differentially expressed in a particular disease state, in a specific way. Lectin affinity chromatography, as it was performed here, has been previously shown to be a repeatable enrichment technique for a comparison of multiple samples that had been individually enriched and quantified in separate label-free proteomic experiments [25]. To illustrate this unique benefit, we have utilized two lectins (AAL and LTA), in a serial fashion, to enrich fucosylated glycoproteins, which have repeatedly been implicated in the progression of several different types of cancers [6,[10][11].…”

Glycoproteomics: Past, present and future

“…Lectins [29] and hydrophilic affinity gels [30] are useful tools for glycoprotein/glycopeptide enrichment. Lectins with relatively broad specificity such as concanavalin A (conA), which recognises high mannose, hybrid and biantennary complex-type N-glycans, are showing considerable promise for enriching serum glycoproteins [31], whilst more specific lectins such as as Vicia villosa lectin (VVL), which preferentially binds to alpha-or beta-linked terminal GalNAc, are valuable tools for purifying glycoproteins that carry the cognate structure. For example bovine pregnancy associated glycoproteins which are rich in glycans carrying the Sda epitope (NeuAcα2-3(GalNAcβ1-4) Gal-) can be efficiently purified from placental tissue using VVL affinity columns [32].…”