Glycoprotein Enrichment Through Lectin Affinity Techniques

Abstract

Posttranslational modifications (PTM) of proteins are among the key biological regulators of function, activity, localization, and interaction. The fact that no more than 30,000-50,000 proteins are encoded by the human genome underlines the importance of posttranslational modifications in modulating the activities and functions of proteins in health and disease. With approximately 50% of all proteins now considered to be glycosylated, its physiological importance in mammalian systems is imperative. Aberrant glycosylation has now been recognized as an attribute of many mammalian diseases, including hereditary disorders, immune deficiencies, neurodegenerative diseases, cardiovascular conditions, and cancer. As many potential disease biomarkers may be glycoproteins present in only minute quantities in tissue extracts and physiological fluids, glycoprotein isolation and enrichment may be critical in a search for such biomarkers. For decades, efforts have been focused on the development of glycoprotein enrichment from complex biological samples. Logically, the great majority of these enrichment methodologies rely on the use of immobilized lectins, which permit selective enrichment of the pools of glycoproteins for proteomic/glycomic studies. In this chapter, lectin affinity chromatography in different formats are described, including tubes; packed columns, and microfluidic channels.

Targeted High-Throughput Glycoproteomics for Glyco-Biomarker Discovery

“…Lectin affinity chromatography can be performed in different formats including tubes, packed columns, microfluidic channels and high pressure liquid chromatography (HPLC) (Mechref, Madera, and Novotny 2008). Different types of support matrices can be used to immobilize the lectins, such as sepharose/agarose beads (Kobata and Endo 1992;Mechref, Madera, and Novotny 2008), magnetic beads (Lin et al 2008), silica or styrene-divinylbenzene co-polymers coated with a cross-linked polyhydroxylated polymer (POROS) (Tousi, Hancock, and Hincapie 2011). Commonly used lectins include mannose and glucose binding concanavalin A (ConA) and N-acetylglucosamine binding wheat germ agglutinin (WGA) for their broad binding specificities and affinity to most Nlinked glycans in biological material.…”

Nutriproteomics: technologies and applications for identification and quantification of biomarkers and ingredients

“…Current MS-based proteomic platforms can deliver a dynamic range of 10 4 . This means that the low-abundant proteome has to be addressed by depletion of the most abundant proteins (15) or by selective enrichment of low-abundant proteins (16)(17)(18) . After protein depletion and/or enrichment, further separation is performed at protein and/or peptide level, based on two-dimensional (2D) gels or on liquid chromatography (LC) or on hybrid approaches (Gel-LC).…”

Role of proteomics in nutrigenomics and nutrigenetics

“…Current mass spectrometry (MS)-based proteomic platforms can deliver a dynamic range of 10 4 . This means that the remaining, as such inaccessible, low-abundance proteome has to be addressed by either depletion of the most-abundant proteins (e.g., by the commercially available multiple affinity removal system that specifically removes the top seven or even 14 plasma proteins) [17], or by selective enrichment of low-abundance proteins (e.g., by the immobilized metal affinity chromatography or titanium dioxide techniques for phosphoproteins [18], lectins [19] or the cell-surface capture technique for glycoproteins [20]). Another way of dealing with the dynamic range is 'focus with a gain in depth at the expense of breadth', and this is based on targeted MS analysis as discussed in the following sections.…”