Abstract
Cassava plays an important role as a staple food for more than 800 million people in the world due to its ability to maintain relatively high productivity even in nutrient-depleted soils. Even though cassava has been the focus of several breeding programs and has become a strong focus of research in the last few years, relatively little is currently known about its metabolism and metabolic composition in different tissues. In this article, the absolute content of sugars, organic acids, amino acids, phosphorylated intermediates, minerals, starch, carotenoids, chlorophylls, tocopherols, and total protein as well as starch quality is described based on multiple analytical techniques, with protocols specifically adjusted for material from different cassava tissues. Moreover, quantification of secondary metabolites relative to internal standards is presented using both non-targeted and targeted metabolomics approaches. The protocols have also been adjusted to apply to freeze-dried material in order to allow processing of field harvest samples that typically will require long-distance transport. © 2019 The Authors. Basic Protocol 1: Metabolic profiling by gas chromatography-mass spectrometry (GC-MS) Support Protocol 1: Preparation of freeze-dried cassava material Support Protocol 2: Preparation of standard compound mixtures for absolute quantification of metabolites by GC-MS Support Protocol 3: Preparation of retention-time standard mixture Basic Protocol 2: Determination of organic acids and phosphorylated intermediates by ion chromatography-mass spectrometry (IC-MS) Support Protocol 4: Preparation of standards and recovery experimental procedure Basic Protocol 3: Determination of soluble sugars, starch, and free amino acids Alternate Protocol: Determination of soluble sugars and starch Basic Protocol 4: Determination of anions Basic Protocol 5: Determination of elements Basic Protocol 6: Determination of total protein Basic Protocol 7: Determination of non-targeted and targeted secondary metabolites Basic Protocol 8: Determination of carotenoids, chlorophylls, and tocopherol Basic Protocol 9: Determination of starch quality.
Highlights
Metabolomics is a constant evolving technology for biochemical analysis of complex mixtures, with the current aim of allowing unbiased, quantitative analysis of very large numbers of metabolites, if not all, in a biological-material extract (Hall & Hardy, 2012)
Metabolites in plants can be separated into primary and secondary/specialized metabolites (Fernie & Pichersky, 2015). Primary metabolites, such as sugars, amino acids, and organic acids, serve as intermediates required for anabolic and catabolic energy metabolism and are found in most free-living plants
PREPARATION OF STANDARD COMPOUND MIXTURES FOR ABSOLUTE QUANTIFICATION OF METABOLITES BY gas chromatography–mass spectrometry (GC-MS) Metabolite profiles are in general expressed as ratios, with data compared with a control sample
Summary
Metabolomics is a constant evolving technology for biochemical analysis of complex mixtures, with the current aim of allowing unbiased, quantitative analysis of very large numbers of metabolites, if not all, in a biological-material extract (Hall & Hardy, 2012). Metabolic profiling using GC-MS allows high accuracy and sensitivity in the analysis of highly complex mixtures of compounds, such as plant tissue samples, permitting identification and robust quantification of metabolites from a single extract. Metabolite profiles are in general expressed as ratios, with data compared with a control sample This protocol can be used for absolute quantification when authentic standards with different amounts of each compound are included in each batch of samples. Place dry aliquots of samples as well as blanks and standard compound mixtures stored at −80°C in a vacuum concentrator or air-flow sample concentrator for 30 min before derivatization. PREPARATION OF STANDARD COMPOUND MIXTURES FOR ABSOLUTE QUANTIFICATION OF METABOLITES BY GC-MS Metabolite profiles are in general expressed as ratios, with data compared with a control sample. C9H18O2 C10H20O2 C11H22O2 C13H26O2 C15H30O2 C17H34O2 C19H38O2 C21H42O2 C23H46O2 C25H50O2
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