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Method development for identification of N-linked glycans by high performance anion exchange chromatography with pulsed amperometric detection and time of flight mass spectrometry
Linköping University, Department of Physics, Chemistry and Biology.
2011 (English)Independent thesis Basic level (university diploma), 10,5 credits / 16 HE creditsStudent thesis
Abstract [en]

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.

 

Place, publisher, year, edition, pages
2011. , 30 p.
Keyword [en]
N-linked glycans, HPAEC-PAD, desalting glycans, TOF
National Category
Analytical Chemistry
Identifiers
URN: urn:nbn:se:liu:diva-66232ISRN: LITH-IFM-G-EX--10/2383--SEOAI: oai:DiVA.org:liu-66232DiVA: diva2:402603
Uppsok
Physics, Chemistry, Mathematics
Supervisors
Examiners
Available from: 2011-03-15 Created: 2011-03-08 Last updated: 2011-03-15Bibliographically approved

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