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Optimierung der Probenvorbereitung für die Analyse von N-Glykanen komplexer Proteingemische mittels Kapillar-Gel-Elektrophorese

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http://pubman.mpdl.mpg.de/cone/persons/resource/persons86603

Karmischin,  Peter
Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Max Planck Society;

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Karmischin, P. (2013). Optimierung der Probenvorbereitung für die Analyse von N-Glykanen komplexer Proteingemische mittels Kapillar-Gel-Elektrophorese. Bachelor Thesis, Otto-von-Guericke-Universität, Magdeburg.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0013-87AE-B
Abstract
The analysis and characterization of complex oligosaccharide structures (glycans) - found in and on membranes of mammalian and bacterial cells as well as viruses - received increasing importance in the recent past [1]. Glycosylation is one of the most common posttranslational modifications of proteins and lipids in animal cells and plays a crucial role in numerous fundamental biological processes [2]. In fact, intracellular as well as extracellular signal transduction, immunological processes and proper protein folding cannot be executed correctly without the right glycosylation of the interacting components. Therefore, investigation and exploration of glycosylation is fundamental to understand these processes. Especially the analysis of N-glycosylation of glycoproteins gained a center stage in scientific research and has also key relevance in production of biotechnologically produces vaccines and other pharmaceutical agents as well as food additives. Within this thesis, a capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF) analysis method is described, which provides both high-sensitive and high-resolution protein N-glycosylation analysis. Unfortunately, in order to analyze the N-glycans of single proteins in complex samples such as blood serum, sample preparation in CGE-LIF based N-glycan analysis is very time-consuming, taking about four to five days. To separate the required protein, usually gel electrophoresis is performed. Afterwards, while the glycosylated protein is still in gel, the N-glycans are cleaved enzymatically. Then, the N-glycans have to be extracted from the gel in order to continue the analysis. The focus of this thesis lays on optimization of this specific step of sample preparation, extraction of N-glycans from polyacrylamide gels. In order to optimize this method, several approaches were tested during this thesis. At first, efficiency of the original extraction technique using ultrasonic was analyzed. Then, an alternative extraction technique was introduced and revised for consistency and reliability. This method passes on ultrasonic and uses the organic solvent acetonitrile for N-glycan extraction. By testing different concentrations of acetonitrile as the extraction agent, the method could be optimized even further. It turned out that using pure water as an extraction agent and shortening the extraction time by 60 % grant the exact same N-glycan-yield as regular extraction using ultrasonic. Using this less resource-intensive approach, the sample preparation of CGE-LIF-based N-glycan analysis for complex samples could be reduced by up to one whole day.