Method development for identification of N-linked glycans by high performance anion exchange chromatography with pulsed amperometric detection and time of flight mass spectrometry
In the biopharmaceutical industry, identification of glycans in a glycoprotein is a regulatory requirement and is a part of the characterization of the protein. Glycans are constructed of several monosaccharides linked together. N-linked glycans, which have been studied in this project, are attached to the nitrogen atom in asparagine.
A method for separating N-linked glycans by high performance anion exchange chromatography had already been developed at the department. To develop a method for identification of the N-glycans by mass spectrometry, a desalting method on porous graphitic carbon (PGC) columns was used and optimized resulting in the eluents A (0,05% TFA in ACN:water 5:95 v/v) and B (0,05% TFA in ACN:water 50:50 v/v). Also the sample introduction on the mass spectrometer was optimized and resulted in a sensitive on-line liquid chromatography mass spectrometry (LC-MS) approach which gave mass spectrometric peaks with high signal to noise ratios and with high mass accuracy.
The developed procedure was then successfully used on glycans cleaved from a glycoprotein separated by high performance anion exchange chromatography with pulsed amperometric detector.
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