High-Throughput N-Glycan Analysis with Rapid Magnetic Bead-Based Sample Preparation

Abstract

N-glycan profi ling of therapeutic glycoproteins is essential to ensure the activity and effi cacy of these promising new-generation drugs. The N-linked glycan moieties of these entities highly affect circulation half-life, immunogenicity and receptor-binding activity as well as physicochemical and thermal stability properties. In addition, more than half of the biopharmaceuticals are glycoproteins representing multibillion dollar worldwide business, further emphasizing the importance of their analysis. In the biomedical fi eld, on the other hand, revealing disease-related glycan structure alterations holds the promise of the discovery of new biomarkers for early diagnostics. Therefore, there is a great demand for widely applicable, high-throughput sample preparation and analysis methods for N-glycan profi ling of glycoproteins. One of the newest exciting developments of the fi eld is the magnetic bead based glycoprotein sample preparation technique. A detailed protocol of this method is given in this chapter in conjunction with rapid capillary electrophoresis analysis of the prepared samples by laser induced fl uorescence detection (CE-LIF). N-glycans are digested by the endoglycosidase PNGase F and the released carbohydrates are labeled with the charged fl uorophore dye of aminopyrenetrisulfonate (APTS). Effective glycan capture by magnetic microparticles enabled fast, easily automated sample preparation both in individual (single vial) and 96-well plate formats, including excess dye removal. Rapid separation of APTS labeled IgG glycans is also shown utilizing an optimized CE-LIF protocol.

Imaging Laser-Induced Fluorescence Detection at the Taylor Cone of Electrospray Ionization Mass Spectrometry

“…A 1.0 mg of maltooligosaccharide mixture, 1.0 mg of maltose, and PNGase F released N-glycans from 1.5 mg of human Immunoglobulin G1 (hIgG1) were APTS labeled following the procedure published earlier by Szigeti et al…”

“…Sample Preparation. A 1.0 mg of maltooligosaccharide mixture, 1.0 mg of maltose, and PNGase F released N-glycans from 1.5 mg of human Immunoglobulin G1 (hIgG1) were APTS labeled following the procedure published earlier by Szigeti et al 37 Capillary Electrophoresis. A CESI 8000 Plus High Performance Separation -ESI Module was used for all separations with bare fused silica surface OptiMS capillaries (both from Sciex), employing a porous sprayer that was connected to the mass spectrometer.…”

N-Glycosylation Profiling of Human Blood in Type 2 Diabetes by Capillary Electrophoresis: A Preliminary Study

“…The reaction mixture was incubated in a heating block with an opened cap at 37 • C overnight under a fume hood [35]. After the labeling step, the excess dye was removed by magnetic beads following the Fast Glycan Sample Preparation and Analysis protocol (Sciex) [36,37] and the APTS-labeled glycan samples were analyzed by CE-LIF.…”