High-throughput immunoglobulin G N-glycan characterization using rapid resolution reverse-phase chromatography tandem mass spectrometry
| Autor: | Bradley D. Prater, Heather M. Connelly, Steven L. Cockrill, Qiang Qin |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Glycan
Spectrometry Mass Electrospray Ionization Glycosylation Time Factors Electrospray ionization Biophysics Analytical chemistry Oligosaccharides CHO Cells Tandem mass spectrometry Biochemistry Cricetulus Tandem Mass Spectrometry Desorption Cricetinae Animals Sample preparation ortho-Aminobenzoates Molecular Biology Chromatography High Pressure Liquid Chromatography Ion exchange Tandem biology Chemistry Cell Biology Reversed-phase chromatography Recombinant Proteins Immunoglobulin G biology.protein |
| Zdroj: | Analytical biochemistry. 385(1) |
| ISSN: | 1096-0309 |
| Popis: | We present an optimized high-throughput method for the characterization of 2-aminobenzamide (2-AB)-labeled N -glycans from recombinant immunoglobulin G (rIgG). This method includes an optimized sample preparation protocol involving microwave-assisted deglycosylation in conjunction with an automated sample cleanup strategy and a rapid resolution reverse-phase high-performance liquid chromatography (RRRP–HPLC) separation of labeled N -glycans. The RRRP–HPLC method permits generation of a comprehensive glycan profile using fluorescence detection in 45 min. In addition, the profiling method is directly compatible with electrospray ionization mass spectrometry (ESI–MS), allowing immediate and sensitive characterization of the glycan moiety by intact MS and tandem MS (MS/MS) fragmentation. We conservatively estimate an efficiency gain of fourfold with respect to the throughput capabilities of this optimized method as compared with traditional protocols (overnight deglycosylation, sample cleanup by graphitized carbon or cellulose cartridge, high-pH anion exchange chromatography, fraction collection, and processing for matrix-assisted laser desorption/ionization time-of-flight [MALDI–TOF] MS analysis) for a single sample. Even greater gains are achieved when processing of multiple samples is considered. |
| Databáze: | OpenAIRE |
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