Identification and Profiling of Auxin and Auxin Metabolites
During the last 10 years, the analytical techniques used in different areas of “life science” have improved tremendously. Mass spectrometry (MS) has become the most versatile and sensitive technique available for identifying and quantifying organic molecu
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Identification and Profiling of Auxin and Auxin Metabolites Ondrˇej Nova´k, Alesˇ Peˇncˇ´ık, and Karin Ljung
Abstract During the last 10 years, the analytical techniques used in different areas of “life science” have improved tremendously. Mass spectrometry (MS) has become the most versatile and sensitive technique available for identifying and quantifying organic molecules, and liquid chromatography-mass spectrometry is the modern analytical tool of choice for analyzing samples of plant, animal and human origin. Both the sensitivity and the selectivity of the available techniques have increased immensely; modern instruments are much smaller, more userfriendly and more versatile than before, and the overall cost of the method has been greatly reduced. However, the required equipment is not available to most plant research laboratories, and most researchers in biology have limited experience with MS techniques. In this chapter, we aim to explain the advantages and limitations of these techniques, and how they can be used in plant research today. More specifically, we demonstrate how different MS techniques can be used for auxin metabolite identification, quantification and profiling. Efficient sample extraction and purification is essential for highly sensitive and selective analyses. We therefore describe selected novel approaches that have been developed to increase the sensitivity of these analyses and make them applicable at the tissue and cellular levels. We also discuss how these techniques can be combined with isotope labelling and mutant analyses to get a better understanding of the metabolic pathways involved in auxin biosynthesis and degradation. Finally, we examine O. Nova´k Umea˚ Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83, Umea˚, Sweden Laboratory of Growth Regulators, Centre of Region Hana´ for Biotechnological and Agricultural Research, Institute of Experimental Botany ASCR and Palacky´ University, Sˇlechtitelu˚ 11, 783 71, Olomouc, Czech Republic A. Peˇncˇ´ık • K. Ljung (*) Umea˚ Plant Science Centre, Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 901 83, Umea˚, Sweden e-mail: [email protected] E. Zazˇ´ımalova´ et al. (eds.), Auxin and Its Role in Plant Development, DOI 10.1007/978-3-7091-1526-8_3, © Springer-Verlag Wien 2014
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the future prospects for the use of MS and other analytical techniques in auxin research as well as the potential for combining these techniques to obtain more information from single samples, and perhaps even from single cells.
1 Extraction of IAA Metabolites from Plant Tissues In general, the isolation and/or pre-concentration of natural bioactive compounds, such as phytohormones, involves multiple critical and often time-consuming steps based on extracting and purifying analytes from a complex plant matrix. Conventional sample preparation methods involve the use of protein precipitation (PP), liquid/liquid extractio
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