nES GEMMA Analysis of Lectins and Their Interactions with Glycoproteins – Separation, Detection, and Sampling of Noncovalent Biospecific Complexes (original) (raw)
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Molecular Pharmaceutics, 2012
With the recent growth of the global monoclonal antibody market, ultrasensitive techniques are required for rapid analysis of possible immunogenic residues, such as galactose-α-1,3-galactose (α-1,3-Gal) on therapeutic proteins expressed in murine or CHO cell lines. We report a capillary electrophoretic approach in conjunction with exoglycosidase digestion for structural elucidation of N-linked IgG glycans containing the above immunogenic epitope. The method uses commercially available reagents and instrumentation, thus making the described methodology readily available for implementation and validation within the biotechnology industry. The method was first evaluated using polyclonal mouse IgG N-glycans which are known to contain α-1,3-Gal containing epitopes. High reproducibility in migration time enabled determination of GU values for five α-1,3-Gal containing structures. The method was successfully applied to the analysis of a NCI reference standard monoclonal antibody and two development phase monoclonal antibodies. The limit of detection and limit of quantitation were 1 and 2 µg of intact protein IgG starting material, respectively, further indicating the high sensitivity of the described method.
Journal of the American Society for Mass Spectrometry, 2018
Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope pepti...
Analytical and Bioanalytical Chemistry
Due to the constant search for reliable methods to investigate glycoproteins in complex biological samples, an alternative approach combining affinity enrichment with rapid and sensitive analysis on-a-chip is presented. Glycoproteins were specifically captured by lectin-coated magnetic beads, eluted by competitive sugars, and investigated with microchip capillary gel electrophoresis (MCGE), i.e., CGE-on-a-chip. We compared our results to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) data, which turned out to be in very good agreement. While SDS-PAGE offers the possibility of subsequent mass spectrometric analysis of captured and separated analytes, MCGE scores with time savings, higher throughput, and lower sample consumption as well as quality control (QC) and process analytical technology (PAT) applicability. Due to these advantages, a lectin-based glycoprotein capture protocol can easily be optimized. In our case, two different types of magnetic beads were tested and compared regarding lectin binding. The selectivity of our strategy was demonstrated with a set of model glycoproteins, as well as with human serum and serum depleted from high-abundance proteins. The specificity of the capturing method was investigated revealing to a certain degree an unspecific binding between each sample and the beads themselves, which has to be considered for any specific enrichment and data interpretation. In addition, two glycoproteins from Trichoderma atroviride, a fungus with mycoparasitic activity and only barely studied glycoproteome, were enriched by means of a lectin and so identified for the first time.
Label-Free Impedimetric Detection of Glycan−Lectin Interactions
Analytical Chemistry, 2007
A compact biosensor for a label-free, rapid (<80 s) detection of glycan-lectin interactions using ac impedance measurements was developed for the first time. A galactose-binding peanut agglutinin (PNA) and sialic acidbinding Sambucus nigra agglutinin (SNA) were covalently surface-immobilized on the layered Cu/Ni/Au printed circuit board (PCB) electrodes. Samples of artificial and natural glycoconjugates consisting of (1) gold glyconanoparticles encapsulated with ∼90-100 copies of TF-antigen disaccharide Galr1-3GalNAc (TF-AuNP), (2) asialofetuin (ASF) containing both LacNAc (Gal 1-4GlcNAc) and TF-antigen, and (3) fetuin (FET), the sialylated glycoform of ASF. The samples were run separately on PNA-and SNA-immobilized PCB electrodes. Our results indicate that TF-AuNP could be rapidly and reliably detected up to 1 pg/mL (13 fM) concentration on PNA electrode but, as expected, yielded no response on the SNA electrode. ASF and FET glycocoproteins were unambiguously detectable up to 10 pg/mL (150 fM) on PNA and SNA electrodes, respectively. Moreover, the technique allowed us to observe glyco-microheterogeneity of FET as well as establish the presence of two isoforms of SNA lectin, SNA-I and SNA-II, in one of the vendor's formulations. Further elaboration of the described technology into novel electrochemically driven lectin arrays may find applications in diagnosis of cancer and other diseases with multiple glycobiomarkers or as a rapid lowcost bioanalytical tool for glycoproteome analyses.
Resolution of Glycoproteins by a Lectin Gel-Shift Assay
Analytical Biochemistry, 2000
Gel-shift assays previously described in the literature are based on protein-protein or protein-DNA interactions. We show that carbohydrate-lectin interactions can be successfully used to alter the electrophoretic mobility of glycosylated, but not nonglycosylated, protein species in SDS-polyacrylamide gels. We were able to separate the two closely migrating mono-(95 kDa) and nonglycosylated (92 kDa) forms of a polytopic membrane protein, anion exchanger 1 (AE1), synthesized by cell-free translation or in transfected HEK293 cells. Concanavalin A was selected as the lectin due to the high mannose content of the oligosaccharide chain on AE1. Concanavalin A was either added to the samples prior to loading or copolymerized in a top layer of the separating gel, the latter being the method of choice. The presence of concanavalin A resulted in slower mobility of the monoglycosylated protein while the mobility of the nonglycosylated form was not altered. The shift in mobility was dependent on concentration of concanavalin A and the length of separating gel containing copolymerized concanavalin A. When a diglycosylated mutant of AE1 was tested, good separation was achieved at lower concentrations of concanavalin A. This lectin gel-shift assay allows the separation of Nglycosylated and nonglycosylated forms of the protein.
Biochemistry, 1977
The effects of several commonly used detergents on the saccharide-binding activities of lectins were investigated using lectin-mediated agglutination of formalin-fixed erythrocytes and affinity chromatography of glycoproteins on columns of lectins immobilized on polyacrylic hydrazide-Sepharose. In the hemagglutination assays, Ricinus communis I (RCAI) and IZ (RCAII), concanavalin A (Con A), and the agglutinins from peanut (PNA), soybean (SBA), wheat germ (WGA), and Limulus polyphemus (LPA) were tested with several concentrations of zwitterionic, cationic, anionic, and nonionic detergents. It was found that increasing detergent concentrations eventually affected hemagglutination titers in both test and control samples, and the highest detergent concentrations not affecting lectin hemagglutinating activities were determined. The effects of detergents on specific binding of [3H]fetuin and a~ialo[~H]fetuin to and elution from columns T h e study of cell surface membrane glycoproteins has been facilitated by the use of lectins which bind saccharides in a highly specific manner . Thus, the number and distribution of surface glycoproteins have been studied by employing radioactive, fluorescent, or electron-dense lectin derivatives . For the study of the chemical nature of membrane glycoproteins, it is desirable to obtain them in a pure form. This is a complicated endeavor, because in the cell membranes that have been well characterized, glycoproteins are classified as integral membrane components ; that is, they are presumed to be stabilized by hydrophobic forces and are not easily solubilized and separated from membrane lipids into aqueous, low ionic strength, neutral solutions. The use of chaotropic agents or detergents (for review,
Nature Biotechnology, 2003
We describe here a strategy for the large-scale identification of N-glycosylated proteins from a complex biological sample. The approach, termed isotope-coded glycosylation-site-specific tagging (IGOT), is based on the lectin column-mediated affinity capture of a set of glycopeptides generated by tryptic digestion of protein mixtures, followed by peptide-N-glycosidase-mediated incorporation of a stable isotope tag, 18 O, specifically into the N-glycosylation site. The 18 O-tagged peptides are then identified by multi-dimensional liquid chromatography-mass spectrometry (LC-MS)-based technology. The application of this method to the characterization of N-linked high-mannose and/or hybrid-type glycoproteins from an extract of Caenorhabditis elegans proteins allowed the identification of 250 glycoproteins, including 83 putative transmembrane proteins, with the simultaneous determination of 400 unique N-glycosylation sites. Because the method is applicable to the systematic identification of a wide range of glycoproteins, it should facilitate basic glycobiology research and may be useful for diagnostic applications, such as genome-wide screening for disease-related glycoproteins.