Biotinylation and high affinity avidin capture as a strategy for LC-MS based metabolomics
Independent thesis Basic level (university diploma), 10 credits / 15 HE creditsStudent thesis
Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the metabolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification.
The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small molecules purification. Specifically, this investigation aims to see if it is achievable to make a biotinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis.
Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermidine, histamine and nicotinamide have been selected.
The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control nonbiotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.
Place, publisher, year, edition, pages
2010. , 52 p.
Biotinylation, avidin-biotin system, metabolites, metabolomics, LC-MS
IdentifiersURN: urn:nbn:se:liu:diva-56771ISRN: LITH-IFM-G-EX--10/2260--SEOAI: oai:DiVA.org:liu-56771DiVA: diva2:322011
2010-06-03, Linköping, 08:45 (Swedish)
UppsokPhysics, Chemistry, Mathematics