Most analytical methods in metabolomics are based on one of two strategies. The first strategy is aimed at specifically analysing a limited number of known metabolites or compound classes. Alternatively, an unbiased approach can be used for profiling as many features as possible in a given metabolome without prior knowledge of the identity of these features. Using high-resolution mass spectrometry with instruments capable of measuring m/z ratios with sufficiently low mass measurement uncertainties and simultaneous high scan speeds, it is possible to combine these two strategies, allowing unbiased profiling of biological samples and targeted analysis of specific compounds at the same time without compromises. Such high mass accuracy and mass resolving power reduces the number of candidate metabolites occupying the same retention time and m/z ratio space to a minimum. In this study, we demonstrate how targeted analysis of phospholipids as well as unbiased profiling is achievable using a benchtop orbitrap instrument after high-speed reversedphase chromatography. The ability to apply both strategies in one experiment is an important step forward in comprehensive analysis of the metabolome.

Anthocyanins in extracts from raspberries and blueberries were analyzed by reversed-phase HPLC coupled to a high-resolution Exactive Orbitrap mass spectrometer (HR-MS) with a resolution of 100,000, operated with an electrospray source in the positive ionization mode. As consumption of anthocyanin-rich berry extracts has been associated with improved cognitive function, brain extracts from European greenfinches ( Carduelis chloris ) that had been fed one blackberry daily for a period of 2 weeks were analyzed by both HPLC with traditional tandem MS in the selected reaction monitoring mode and HPLC-HR-MS. Cyanidin-3-O-glucoside was detected in the brain extracts by both methods, but because of its high level of selectivity, HR-MS was ca. 200-fold more sensitive. A further advantage of HR-MS is that unlike MS-SRM it enables both targeted and nontargeted compounds to be detected and much lower limits of detection are achieved without compromising the selectivity of the analysis.

Plant cell wall polysaccharides vary in quantity and structure between different organs and during development. However, quantitative analysis of individual polysaccharides remains challenging, and relatively little is known about any such variation in polysaccharides in organs of the model plant Arabidopsis thaliana. We have analysed plant cell wall pectic polysaccharides using polysaccharide analysis by carbohydrate gel electrophoresis. By highly specific enzymatic digestion of a polysaccharide in a cell wall preparation, a unique fingerprint of short oligosaccharides was produced. These oligosaccharides gave quantitative and structural information on the original polysaccharide chain. We analysed enzyme-accessible polygalacturonan (PGA), linear beta(1,4) galactan and linear alpha(1,5) arabinan in several organs of Arabidopsis: roots, young leaves, old leaves, lower and upper inflorescence stems, seeds and callus. We found that this PGA constitutes a high proportion of cell wall material (CWM), up to 15% depending on the organ. In all organs, between 60 and 80% of the PGA was highly esterified in a blockwise fashion, and surprisingly, dispersely esterified PGA was hardly detected. We found enzyme-accessible linear galactan and arabinan are both present as a minor polysaccharide in all the organs. The amount of galactan ranged from ~0.04 to 0.25% of CWM, and linear arabinan constituted between 0.015 and 0.1%. Higher levels of galactan correlated with expanding tissues, supporting the hypothesis that this polysaccharide is involved in wall extension. We show by analysis of mur4 that the methods and results presented here also provide a basis for studies of pectic polysaccharides in Arabidopsis mutants.

No abstract