Nontargeted Metabolome Analysis by Use of Fourier Transform Ion Cyclotron Mass Spectrometry

Abstract

Advanced functional genomic tools now allow the parallel and high-throughput analyses of gene and protein expression. Although this information is crucial to our understanding of gene function, it offers insufficient insight into phenotypic changes associated with metabolism. Here we introduce a high-capacity Fourier Transform Ion Cyclotron Mass Spectrometry (FTMS)-based method, capable of nontargeted metabolic analysis and suitable for rapid screening of similarities and dissimilarities in large collections of biological samples (e.g., plant mutant populations). Separation of the metabolites was achieved solely by ultra-high mass resolution; Identification of the putative metabolite or class of metabolites to which it belongs was achieved by determining the elemental composition of the metabolite based upon the accurate mass determination; and relative quantitation was achieved by comparing the absolute intensities of each mass using internal calibration. Crude plant extracts were introduced via direct (continuous flow) injection and ionized by either electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) in both positive or negative ionization modes. We first analyzed four consecutive stages of strawberry fruit development and identified changes in the levels of a large range of masses corresponding to known fruit metabolites. The data also revealed novel information on the metabolic transition from immature to ripe fruit. In another set of experiments, the method was used to track changes in metabolic profiles of tobacco flowers overexpressing a strawberry MYB transcription factor and altered in petal color. Only nine masses appeared different between transgenic and control plants, among which was the mass corresponding to cyanidin-3-rhamnoglucoside, the main flower pigment. The results demonstrate the feasibility and utility of the FTMS approach for a nontargeted and rapid metabolic "fingerprinting," which will greatly speed up current efforts to study the metabolome and derive gene function in any biological system.

Accurate mass error correction in liquid chromatography time-of-flight mass spectrometry based metabolomics

“…Technical advances in mass spectrometry, resulting in increased mass resolution and accuracy, enable the (putative) identification of metabolites simply by their accurate mass determinations (Aharoni et al 2002;Lim et al 2007;Murch et al 2004;Thurman et al 2005). For instance, Fourier transform ion cyclotron mass spectrometry (cyclotron FTMS) and Orbitrap FTMS (Olsen et al 2005) typically analyse the masses of ions with an ultra high accuracy of less than 1 ppm deviation from the calculated masses.…”

Rice Metabolomics

“…Extraction performed twice, i.e., once each with polar and non-polar buffers, for one sample is surely considered to be more effective to encompass many metabolites, as compared to extraction at one time with one buffer, although the time and labor for the analysis of each sample are doubled. In this case, hydrophilic and hydrophobic buffers such as 50% methanol and 100% acetonitrile are used [1]. Second, the sample preparation step must be taken into account.…”

“…Therefore, various separation and detection techniques, such as GC-MS [31,65], liquid chromatography (LC)-MS [62,70], CE-MS [83,92], Fourier transform ion cyclotron resonance (FT-ICR) MS [1,73,75] and NMR [61,85] have been applied to metabolomics. Among them, GC-MS is the most popular method in current metabolomic studies [31].…”

An introduction to liquid chromatography–mass spectrometry instrumentation applied in plant metabolomic analyses

“…DIMS provides a rapid (∼2 min per sample) metabolite fi ngerprint (Goodacre et al, 2003;Allwood et al, 2006), which has proven to be valuable for a fi rstround screen of material (indicating robust sample growth, collection and extraction methods) prior to more expensive and time-consuming chromatography combined with MS approaches (Catchpole et al, 2005). DIMS is also gaining in popularity with the advent of high-resolution and -precision Fourier transform ion cyclotron resonance (FT-ICR)-MS instruments (Aharoni et al, 2002;Hirai et al, 2004;Southam et al, 2007), although without the application of chromatography, DIMS alone cannot resolve isobaric metabolites. GC-MS has proven to be a robust and reproducible tool for the study of volatile organic compounds (VOCs; Li et al, 2006Li et al, , 2008Zhang and Li, 2007;Riazanskaia et al, 2008) as well as a range of largely primary metabolites made amenable to GC-MS detection via the addition of volatile trimethylsyl (TMS) groups by N-methyl-N-trifl uoroacetamide (MSTFA) based derivatisation (Fiehn et al, 2000;Fernie et al, 2004;Lisec et al, 2006;Biais et al, 2009;Allwood et al 2009).…”