April 1, 2007 (Vol. 27, No. 7)
Peter Porschewski Ph.D.
Thibault Geoui Ph.D.
Qproteome GlycoArray Kit for Determining the Pattern & Abundance of Glycosylation Epitopes
Glycosylation is the most versatile and one of the most abundant of all co- and post-translational modifications. It results from the addition of sugar residues to a protein backbone to form a glycoprotein. Glycosylation plays an important role in many biological functions, including immune defense, fertilization, viral replication, parasitic infection, cell growth, inflammation, and cell-cell adhesion.
In vivo, glycosylation is tissue-dependent and can vary significantly with cell state. Proteins are glycosylated by the actions of a series of glycosidases and glycosyltransferases that act sequentially on the growing glycan as it passes through the lumen of the endoplasmic reticulum and the Golgi apparatus. Since the various enzymatic reactions may not all reach completion, a variety of glycan structures are commonly attached at each glycosylation site. Consequently, under a given set of conditions, different populations of glycosylation forms (glycoforms) may be generated for a single protein.
The number of glycoforms and their relative abundance within a cell are affected by the intrinsic structural properties of the individual protein, as well as the repertoire of glycosylation enzymes available (including their type, concentration, kinetic characteristics, compartmentalization). This repertoire has been shown to change upon changes in cell state (e.g., oncogenic transformation).
In vitro, glycosylation strongly depends on growth conditions—e.g., type of cell, nutrient concentrations, pH, cell density, and age of culture—all of which affect the glycosylation patterns of glycoproteins.
Characterizing Recombinant Glycoprotein from Cell Cultures
By providing researchers with a simple, rapid, kit-based method for determining the pattern and relative abundance of specific mammalian glycosylation epitopes, the Qproteome GlycoArray Kit from Qiagen (www.qiagen.com) can be used as a first-line tool for the gross characterization of recombinant glycoproteins. The technology consists of arrays of selected lectins, which are used to determine the glycosylation features of the analyzed glycoproteins.
Lectins are a family of carbohydrate-recognizing proteins that are classified into a number of specificity groups based on the monosaccharides for which they exhibit the highest affinity. The arrays in the kit contain over 20 lectins with overlapping specificities, which have been characterized using a large dataset of carefully chosen, well-characterized glycoproteins. The different lectins on the array bind specific glycan features. By locating the positions where a protein has bound, using either labeled protein or a fluorescent antibody, the protein’s glycan makeup can be derived.
The analysis delivers a “fingerprint” of the protein’s glycan makeup, which can be deconvoluted to provide information on both the presence and relative abundance of specific glycan features and can be used as a reference to compare protein batches. The analysis can be performed on crude samples in growth media, eliminating the need for time-consuming purification and sample-preparation steps.
Principles and Procedure
Lectins with differing, defined glycan-binding specificities are spotted on the surface of an array. The array is probed with a glycoprotein, washed to reduce background, and visualized using a microarray scanner. Bound glycoproteins can be visualized by direct fluorescent labeling of the glycoprotein target(s), biotinylation of the target(s) followed by detection with fluorescent streptavidin, or by using a fluorescently labeled protein-specific antibody. After scanning the array, fluorescent signals are evaluated using Qproteome GlycoArray Analysis Software (Figure 1).
By providing a comprehensive analysis of glycan makeup, the Qproteome GlycoArray Kit can be used for glycoprotein studies in the fields of clone screening, selection, and optimization; ADME and toxicology; stability testing; and QA/QC and batch monitoring.
Qproteome GlycoArrays provide researchers with a simple kit-based method for characterizing the glycosylation pattern of recombinant proteins. The method is more rapid than conventional methods that rely on chromatographic and mass spectrometric techniques, as there is no need for purification and time-consuming sample-preparation steps.
Peter Porschewski, Ph.D., is a scientist in the protein purification and assays department and Thibault Geoui, Ph.D., is a marketing specialist for proteins at Qiagen. Web: www.qiagen.com. Phone +49 2103 29 16270. Email: [email protected].