Article pubs.acs.org/JAFC
Comprehensive Multiphase NMR Spectroscopy of Intact Seeds
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C‑Labeled
Leayen Lam,†,‡ Ronald Soong,†,‡ Andre Sutrisno,†,‡ Ries de Visser,§ Myrna J. Simpson,†,‡ Heather L. Wheeler,#,⊥ Malcolm Campbell,#,⊥ Werner E. Maas,⊗ Michael Fey,⊗ Antonie Gorissen,§ Howard Hutchins,⊗ Brian Andrew,⊗ Jochem Struppe,⊗ Sridevi Krishnamurthy,⊗ Rajeev Kumar,○ Martine Monette,○ Henry J. Stronks,○ Alan Hume,○ and André J. Simpson*,†,‡ †
Department of Physical and Environment Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario, Canada M1C 1A4 ‡ Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario, Canada M5S 3H6 § IsoLife BV, Droevendaalsesteeg 1, 6708 PB Wageningen, The Netherlands # Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto, Ontario, Canada M1C 1A4 ⊥ Department of Cell Systems and Biology, University of Toronto, 33 Willcocks Street, Toronto, Ontario, Canada M5S 3B2 ⊗ Bruker BioSpin Corp., 15 Fortune Drive, Billerica, Massachusetts 01821-3991, United States ○ Bruker BioSpin Canada, 555 Steeles Avenue East, Milton, Ontario, Canada L9T 1Y6 S Supporting Information *
ABSTRACT: Seeds are complex entities composed of liquids, gels, and solids. NMR spectroscopy is a powerful tool for studying molecular structure but has evolved into two fields, solution and solid state. Comprehensive multiphase (CMP) NMR spectroscopy is capable of liquid-, gel-, and solid-state experiments for studying intact samples where all organic components are studied and differentiated in situ. Herein, intact 13C-labeled seeds were studied by a variety of 1D/2D 1H/13C experiments. In the mobile phase, an assortment of metabolites in a single 13C-labeled wheat seed were identified; the gel phase was dominated by triacylglycerides; the semisolid phase was composed largely of carbohydrate biopolymers, and the solid phase was greatly influenced by starchy endosperm signals. Subsequently, the seeds were compared and relative similarities and differences between seed types discussed. This study represents the first application of CMP-NMR to food chemistry and demonstrates its general utility and feasibility for studying intact heterogeneous samples. KEYWORDS: multiphase, broccoli, corn, wheat, seeds, NMR spectroscopy, intact sample, in situ analysis, triacylglyceride (TAG)
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pulse field gradients and a spectrometer lock restrict the type of experiments and information that can be extracted directly. Recently, NMR spectroscopy has been applied in seed analysis for the study of oil and protein composition, but often requires extensive and time-consuming sample preparation.8,9 One study used wheat seeds that were initially milled into a flour, dissolved in a buffer solution, and centrifuged, and the supernatant was collected prior to NMR analysis.10 Such methods of sample preparation can be detrimental as they potentially perturb the structure and native chemical and physical interactions that influence analyte kinetics across phase boundaries, which are important for analysis. The first 1H measurements of intact seeds, in which the oil content was determined for a variety of seeds, were likely performed in 1963.11 This was followed by the first highresolution 13C measurements conducted in 1974, whereby the oil composition was measured for a single soybean.12 Previous
INTRODUCTION
Seeds are integral to world nutrition as they not only serve as a direct source of food rich with essential vitamins, fiber, sterols, and antioxidants but carry the potential to be cultivated into fruit- and vegetable-bearing plants.1−4 Plant sterols have been shown to reduce low-density lipoprotein cholesterol absorption, whereby the structurally analogous sterols compete with cholesterol absorption sites in the intestine.5 Similarly, high antioxidant intake has demonstrated protective effects against chronic diseases such as cancer, cardiovascular disease, osteoporosis, and diabetes by mitigating the damaging effects related to oxidative stress.1,2,4 β-Carotene, a precursor to vitamin A, and other antioxidants such as vitamin C are not produced natively by the human body and must be obtained from extrinsic dietary sources, for which fruits and vegetables are naturally abundant.6,7 Historically, NMR spectroscopy has evolved into two separate fields, namely, solution-state and solid-state NMR. Seed components can be extracted for solution-state NMR, but this process is destructive and selective toward only a subset of chemicals present. Solid-state NMR can be used to study intact seeds, but in the case of soluble/gel components, the lack of © 2013 American Chemical Society
Received: Revised: Accepted: Published: 107
October 12, 2013 December 18, 2013 December 19, 2013 December 19, 2013 dx.doi.org/10.1021/jf4045638 | J. Agric. Food Chem. 2014, 62, 107−115
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that are involved in phase changes (drying, swelling), and molecular interactions (for example, between a herbicide and plant tissue) and, thus, has considerable potential for the analysis of food, soil, sediments, plants, and seeds.
analysis of intact seeds has been performed by high-resolution magic angle spinning (HR-MAS) of canola seeds to determine seed oil composition;13 cross-polarization (CP)-MAS and HRMAS to characterize Arabidopsis, pea, and lettuce seeds;14 metabolite profiling to assess conifer seed quality,15 as well as measuring the moisture content of garden cress seeds.16 In all cases, although intact seeds were used, only select phases (liquid, gel, or solid) were studied in a given experiment. Traditional solution-state NMR probes use low-power electronics, a lock channel, and pulsed field gradients and provide excellent line shape but only for dissolved samples. HRMAS probes were introduced in 1996 and employ magic angle spinning, a magic angle gradient, and susceptibility-matched stators.17 HR-MAS probes permit the study of swellable and liquid components. However, HR-MAS probes are designed using low-power circuitry; as such, they cannot generate the RF field required for high-power decoupling or cross-polarization, elements essential to the majority of solid-state NMR experiments. Solid-state probes, on the other hand, are designed to generate high RF fields, but as solid-state NMR spectroscopy has been predominantly reserved for the study of true solids, they lack a lock and gradients, which are required for the efficient study of liquid and gel components. Comprehensive multiphase (CMP) NMR, introduced in 2012, incorporates all of the aforementioned aspects, including magic angle spinning, a magic angle gradient, a lock, full susceptibility matching, and solid-state circuitry to permit high power handling. Therefore, it is built to study unaltered samples where all organic components can be observed and differentiated in situ, resulting in a universal approach.18 The use of separate probes to achieve the same goal is only an option for the most simple, structural studies, where the sample does not change. Even in this case, it is important to stress that in large part, due to independent development of liquid- and solid-state NMR, very few laboratories in the world would have separate liquid, HR-MAS, and solids probes, and even if they did, scheduling all to be available at the same time would be extremely challenging. More importantly, any study involving kinetics transfer between phases (e.g., growth, contaminant sequestration) or changes of one phase into another (for example, soil swelling/drying, feeding phenylalanine to follow lignin formation) will require a CMP probe as such studies are impossible to perform using separate probes. This is discussed in more detail later in this study. CMP-NMR has been, thus far, used to determine the fate and binding of contaminants in soil.18,19 Here, we introduce CMP-NMR to applications in food and agriculture. This study focuses on structural information that can be obtained from 1H and 13C CMP-NMR characterization of intact broccoli, corn, and wheat seeds. Wheat and corn were selected as they represent major global crops, whereas broccoli seeds were included as an example of a legume. The different seed types will be compared to each other for relative differences and similarities as well as further considerations of CMP probes (including quantification; need for labeling and future potential) will be discussed toward the end of this study. Seeds are used here as an example that serves to demonstrate the general applicability of CMP-NMR for the analysis of all organic components in all phases in whole, unaltered samples. CMP-NMR is likely to find widespread application in the agricultural and food sciences due to versatility and ability to provide unsurpassed molecular detail on intact samples. CMPNMR has potential to understand intact structure, processes
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MATERIALS AND METHODS
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C Labeling of the seeds. Uniformly 13C-enriched seeds of broccoli, corn, and wheat (Brassica oleracea var. botrytis ‘Broccoli’, Zea mays, and Triticum aestivum, respectively) were produced in specially designed, airtight, high-irradiance growth chambers20 (IsoLife, Wageningen, The Netherlands). Plants were grown from 13C-labeled seeds in a closed atmosphere containing 97 atom % 13CO2 (from pressurized cylinders; Isotec, Inc., Miamisburg, OH, USA) from the seedling stage until full maturity. Internal wind speed ensured efficient pollination of the corn and wheat. Pollination of broccoli flowers was ensured by combining compatible parent plants and introducing bluebottle flies into the chambers. Mineral nutrients were supplied as Hoagland-type solutions with micronutrients and iron.21,22 Climate conditions were as follows: irradiance (PPFD), 600 μmol m−2 s−1 (HPI) during a 16 h day; day/night temperatures, 24/16 °C; relative humidity, 75/85%. Sample Preparation. Uniformly 13C-labeled (>97% total carbon content) and nonlabeled (80% confidence match (from automated searches) were further selected for detailed manual inspection. Only compounds that showed near perfect matches in all spectral regions were retained as assignments. The chemical shifts of the identified compounds were compared with database values (r2 = 0.99, σ = 0.01). For quantification, spectra were also normalized to total intensity over the 200−0 ppm region in the carbon spectra. The multi-integration tool in AMIX (version 3.9.3, Bruker BioSpin) was used to compare the relative quantity of carbonyl 109
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Figure 2. 13C NMR spectra of a single 13C-labeled wheat seed: (a) components with restricted diffusion (by diffusion editing, DE); (b) semisolids (by recovering relaxation losses arising from diffusion editing, RADE); (c) HSQC with triacylglyceride signals in black and all other signals gray; (d) ACD/Laboratories HSQC simulation of generic triacylglycerides structure (overlaid). protein (defined as 190−170 ppm), carbohydrates (defined as 110−50 ppm), lipids/fatty acids (defined as 183−164, 135−121, and 44−3 ppm), and aliphatic proteins (defined as 60−40 ppm).
1a). In this experiment carbons from true solid components are strongly attenuated as the lower power decoupling is insufficient to decouple broad proton resonances that are characteristic in solids. As such, the spectrum contained signals from components with and without restricted diffusion and semisolids, but signals from true solids are largely suppressed (Figure 1a). Due to the complex nature of the seeds, the conventional 13C spectrum showed considerable overlap, which was reduced by spectral editing to obtain the IDE spectrum (Figure 1b). IDE shows only molecules that demonstrate unrestricted diffusion in solution. It requires the subtraction of the diffusion-edited spectrum (contains molecules with restricted diffusion) from a reference spectrum (without diffusion weighting, i.e., defocusing/refocusing gradients set to zero power) but otherwise acquired under identical conditions. The IDE spectrum (Figure 1b) emphasized a range of small molecules, but identification from the IDE spectrum alone is challenging. Two-dimensional HSQC correlations provided additional spectral dispersion as well as one-bond 1H−13C connectivity information. Database matching against the AMIX Bruker Bioreference spectral database of the HSQC data along with 1H−1H COSY, 1H−1H TOCSY, and 1H−1H NOESY (see Supporting Information Figure S1 for example COSY and TOCSY data) confirmed a wide range of metabolites present (Figure 1c; see Supporting Information Figure S2 for an expansion of Figure 1c and brief description of each metabolite identified). After identification from the 2D spectrum, metabolite assignments were transferred to the 1D carbon spectrum (Figure 1a). Major regions can be summarized as follows: carbonyls from proteins and lipids (approximately 190−170 ppm); guanidine group of arginine (159 ppm); conjugated double bonds (135−130 ppm) from triacylglycerides (TAG, see next section); ethylene (sharp signal at 128.1 ppm); anomeric carbons of carbohydrates (90−110 ppm); various overlapping carbohydrate and amino acid signals (90−50 ppm); methanol (56.40 ppm) and amino acids and various metabolites (42−46 ppm including 4-aminobutyric acid, arginine, cadaverine, lysine, ornithine, putrescine) and aliphatics (50−5 ppm, mainly TAG, see next section). As discussed above, the IDE spectrum (Figure 1b) highlighted the molecules that are most liquid-like or dynamic within the seed. To our knowledge, this is the first report of
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RESULTS AND DISCUSSION Comprehensive Multiphase NMR Spectroscopy. In this study a number of spectral editing approaches have been used. These have been discussed in detail by Courtier-Murias et al.18 Briefly, starting from the most liquid-like through the most solid-like, these experiments can be described as follows. (1) IDE is a difference-based approach that selects molecules that have unrestricted tumbling (i.e., truly dissolved molecules). Here, these components will be referred to as “components with unrestricted diffusion”. (2) DE selects molecules with restricted diffusion and will include swollen biopolymers, mobile gels, and smaller molecules that are trapped or sorbed. In this study these components will be referred to as “components with restricted diffusion”. There is no clear-cut diffusivity that separates all dissolved molecules from all those with restricted diffusion. Instead, the experiments should be considered as a continuum with the “fast diffusing molecules contained in IDE” and generally “the restricted molecules” being in DE. The strength of the diffusion editing has been developed on standard samples to give the best distinction between truly dissolved molecules from entrapped molecules and gels.18 (3) RADE is an experiment that compensates for signals that otherwise may be lost through relaxation during diffusion editing. RADE selects semisolid components that may include gels and possibly some very dynamic solids. In this study these components will be referred to as “semisolids”. (4) T2 filtered CP-MAS selects the more mobile “true solids” that may include very rigid gels and solids that exhibit some dynamics. In this study these components will be referred to as “dynamic solids”. It is important to note that although no components are missed, some components may be observed twice by 1H RADE and 13C T2 CP-MAS.18 (5) Finally, inverse T2 filtered CP-MAS is a difference approach that selects just the truly rigid solids that show little to no dynamics. In this study these components will be referred to as “rigid solids”. Detailed Analysis of Wheat Seed. Components with Unrestricted Diffusion (Soluble Components). A conventional 1D carbon profile of the single 13C-labeled (97 atom %) wheat seed was acquired with low-power waltz-16 decoupling (Figure 110
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ethylene, a gas within the seed,30 detected by NMR spectroscopy. Strong signals from methanol and a range of small sugars dominated, indicating that these are present in a dissolved or dynamic state. Conversely, the aliphatic signals hardly contribute at all to the IDE spectrum and suggest many of these are in a more restricted environment. This will be discussed in the next section. Components with Restricted Diffusion and Semisolid Components via Diffusion-Based Editing. Diffusion editing encodes the spatial position of a signal at the start of the experiment and decodes it at the end. If the molecule physically changes position during the experiment, it is not refocused and is attenuated. The result is a spectrum that contains signals from molecules that show very slow/no diffusion, such as macromolecules, swollen polymers, and small molecules trapped in an environment that prevents free diffusion. Figure 2a shows the 13C DE spectrum of the wheat seed. Small signals from carbohydrate were present, indicating that some carbohydrates were present in a restricted diffusion-like state. These could be swollen carbohydrate polymers (for example, starch) or smaller entities sorbed to other larger components. The dominant signals in the DE spectrum arose from triacylglycerides (TAG). The HSQC of the single 13Clabeled wheat seed (Figure 2c) matches extremely well with the ACD/Laboratories HSQC simulation of the generic TAG structure overlaid (Figure 2d). Whereas generic TAG structures clearly dominate the lipid profile, individual TAG molecules could not be distinguished. Detailed assignments are present in Table 1 and are consistent with COSY, TOCSY, NOESY, and
Murias and co-workers.18 In simple terms, RADE accounts for signals from semisolids that otherwise could be missed by diffusion-based spectral editing alone. Figure 2b shows the RADE spectrum for the 13C-labeled wheat seed. The spectrum contains signals from the components that relax extremely quickly and have a semisolid character. As with the spectrum of components with restricted diffusion, there is a strong signature from TAG suggesting that some of the TAG have a semisolidlike character as expected. Other lipid signals may arise from membrane structures within the seed. In addition to the aliphatic species, the RADE spectrum also contains significant carbohydrate signal, indicating strong contribution from carbohydrate polymers. These could arise from the starchy endosperm of wheat seeds33 as well as cell walls and possibly more flexible components of the seed coat itself. These carbohydrate signals are strongly emphasized by CP-MAS (Figure 3a), which emphasizes the true solid in the seed coat.34 Solid Components. The above experiments focused on the unrestricted diffusion (soluble), restricted diffusion, and semisolid components of 13C-labeled seeds. As CMP-NMR probes can handle the high-power RF requirements required for modern solid-state experiments, the solid components of the seeds can also be studied. CP-MAS is an excellent filter for the 13C detection of true solid components.18 During CP, magnetization is passed from proton on carbon via a strong dipole network. As such, CP is not efficient for dynamic systems (for example, solutions, mobile gels). However, in the true solids static H−C dipoles provide a useful framework for CP and the process is highly efficient. The result is that CP provides a strong bias toward the components in the sample with the most solid-like character. The solid-state NMR spectral profile is shown in Figure 3a and is dominated by starch, cellulose, and hemicellulose signals. Assignments for all peaks have previously been reported14,35 and are labeled in Figure 3a. Absolute quantification is challenging because of overlap of signals, and additional in-depth research is required to understand how the editing procedure affects quantification. It is unclear how the different editing steps influence absolute quantification; however, it is possible to do relative quantification between the species (see next section) to estimate crude changes or differences. T2 relaxation editing can be used to further edit the solid components with dynamics from the true solid domains. The proton T2 filter is utilized prior to CP so that protons in the most solid environment with short T2 will preferentially relax. This leaves only the resonances from the unrestricted diffusion or semimobile materials (Figure 3c). The most rigid signals with short T2 can be recovered with spectral subtraction of the T2-filtered CPMAS spectrum (dynamic solids) from the regular CP-MAS spectrum (everything), leaving a spectrum containing the most rigid resonances (Figure 3b). For example, the peak for amorphous CH2 does appear in T2-filtered CP-MAS (labeled “9” in Figure 3c), demonstrating these chains exhibit motion and likely exist as restricted diffusion/semisolid state. Conversely, the signal labeled “5” (Figure 3b) arose from crystalline cellulose. Specifically, this signal represents the C4 position in crystalline cellulose and is the only signal that can be clearly resolved due to overlap. Crystalline cellulose behaves like a true solid; as such it is strongly emphasized along with the most rigid components (Figure 3b). Other carbohydrate signals, mainly starch, are prominent in all spectra and come from the starchy endosperm of wheat seeds33 and from the seed coat.34 Next to TAG, starch and
Table 1. Proton (1H) and Carbon (13C) Chemical Shift Assignment of Fatty Acid and Lipidic Components of a Single 13C-Labeled (97 Atom %) Wheat Seed component CH3−(CH2)n− CH3−(CH2)n− −(CH2)n−CH2−CH2 −(CH2)n−CH2−CH2 −(CH2)−CH2−CO−O− −CH2−CHCH− −CH2−CH2−CO−O− −CHCH−CH2−CHCH− −CH2O− −CHO− −CH2−CHCH−
1
H (ppm)
0.90 1.31 1.31 1.31 1.59 2.05 2.25 2.76 4.08−4.28 5.21 5.32
13
C (ppm)
16.60 25.30 32.20 34.20 27.60 29.80 36.40 28.20 64.40 71.50 130.90−132.20
the previous work by Sacco and co-workers.31 Complete TAG oxidation yields twice the amount of energy that could be generated from protein or carbohydrates.32 Thus, TAGs are considered highly compact energy reserves and are therefore an efficient way to maximize energy storage within the confines of a seed.32 Most triglycerides exist as a “solid fat” somewhat analogous to butter; as such, it is logical that TAG within the seeds display restricted diffusion-like properties rather than true liquid or solid properties. Diffusion editing is a useful experiment to emphasize components with restricted movement. However, the experiment uses a relatively long diffusion delay, which could potentially lead to the loss of signal from very large or semisolid components that exhibit fast relaxation. This rapidly relaxing signal can be fully recovered using an experiment termed RADE, the technicalities of which are discussed by Courtier111
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at 30 ppm and carbonyl at 130 ppm (not shown) each are not correlated to carbohydrate signals. Comparing Wheat, Broccoli, and Corn Seeds. For unrestricted diffusion (soluble) components (IDE), corn has the most signals in the free carbohydrate area (about 10% more than wheat and 55% more than broccoli, based on integration), with the majority of signals arising from glucose and fructose, which is consistent with the literature (Figure 4c).37,38 It is important to note that in the case of corn, the seed was cut in half, and it is possible that some of this additional signal intensity from soluble molecules could arise from D2O leaching components from the exposed interior of the seed. The IDE (unrestricted diffusion (soluble) components, Figure 4b) and DE (restricted diffusion components, Figure 4e) for broccoli looked quite similar, and both were dominated by lipids in large part because of the lower carbohydrate levels compared to the other two species. Broccoli had the highest lipid content (about 10% more than wheat and corn based on integration), and these findings are consistent with trends found in the literature.39,40 The actual location of these small molecules within the seeds is unclear; previous MRI-based studies have indicated that water has some mobility within the endosperm,41,42 and it is possible that some of these relatively free molecules are associated with this water. The RADE spectrum (semisolids) of corn and wheat (Figure 4i) emphasized a considerable contribution of carbohydrates.40 Conversely, broccoli shows very few carbohydrates in the semisolid phase. Note that in wheat, broccoli, and corn the CH3 signal of lipids (marked with an askterisk (∗) in Figure 4a−f) appeared in the IDE and DE but disappeared in the RADE spectrum. This likely resulted from the local motion of the CH3 terminal groups that leads to longer relaxation times compared with other parts of the lipid/TAG molecules. The CP-MAS of broccoli (Figure 4k) was very different from that of wheat (Figure 4j) mainly because it had stronger signals from proteins (about 2.5 times more intense based on integration) and from long chain fatty acyl groups than the wheat spectrum. This is consistent with the literature that lists protein in broccoli seeds to be ∼21 g/100 g39 and in wheat to be ∼10 g/100 g.40 The 60−0 ppm region mainly arises from aliphatic amino acids with the strong signals from long-chain aliphatics (CH2)n superimposed at ∼30 ppm. α-Carbons from protein resonate in a band from ∼60 to 40 ppm centered at ∼50−55 ppm, and aromatic amino acids add to the aromatic region and are most prominent from 110 to 120 ppm. The carbonyl signals at 190−160 ppm arise from proteins, lignins, lipids, and hemicelluloses. The carbonyls are likely much more intense in the broccoli compared to the wheat (roughly 5.5 times more based on integration) in large part due to the additional protein and oil content.39,40 The presence of a relatively strong (CH2)n superimposed at ∼30 ppm in the broccoli is interesting. This suggests that in the broccoli, at least some of the aliphatic components are more solid-like than in the other seeds. As the characteristic double bonds from TAG are also present (∼125 ppm), it is most likely that the truly solid aliphatic component is a portion of TAG stored in the more solid form when compared to the other seeds. However, it is also possible that some of this additional CH2 intensity also arises from lipoprotein, free fatty acids, or lipids other than TAG, which are known to be present in seeds.15,43,44 Other Considerations. CMP-NMR provides a unique insight into both chemical and physical attributes of molecular structure inside unaltered natural samples. Seeds here were
Figure 3. 13C NMR spectra of a single 13C-labeled wheat seed. (a) True solids (by cross-polarization magic angle spinning, CP-MAS). Peaks: 1, carbonyls in lignins, hemicelluloses, and proteins; 2, double bonds (lignins); 3, C1 of cellulose and hemicelluloses; 4, C1 of starch (anomeric carbon); 5, C4 of crystalline cellulose; 6, C4 of amorphous cellulose, hemicelluloses, and/or starch; 7, C2, C3, C5 in celluloses, hemicelluloses, and starch and C6 starch branch points; 8, C6 in celluloses, hemicelluloses, and starch; 9, amorphous CH2; 10, aliphatic. (b) Spectral editing to emphasize rigid solids. (c) T2-filtered CP-MAS to emphasize dynamic solids. (d) Dipolar assisted rotational resonance (DARR) to highlight connectivities between carbons for the truly solid components.
storage protein are the main storage reserves of cereal seeds. Higher plants produce starches, which are a critical nutritional source for humans.36 DARR is able to identify 13C−13C correlations through space (Figure 3d) and is a very useful experiment to confirm and help assign structure. DARR confirms that the signals at 62−73, 62−100, and 73−100 ppm are correlated in the same structure, consistent with that of carbohydrates in general. DARR also confirms that the TAG 112
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Figure 4. 13C NMR spectra comparing 13C labeled wheat, broccoli, and corn seeds: (a−c) unrestricted diffusion (soluble) components (by inverse diffusion edited, IDE); (d−f) restricted diffusion (by diffusion edited, DE); (g−i) semisolids (by recovering relaxation losses arising from diffusion editing, RADE); (j−l) true solids (by cross-polarization magic angle spinning, CP-MAS). The −CH3 of TAG is marked with an *; 1, dominated by fructose in corn; 2, triacylglycerides; 3, carbonyls (result of increased protein content); 4, aromatic (result of increased protein content); 5, α carbon of amino acids; 6, dominated by aliphatic amino acids; 7, aliphatic −(CH2)− (dominated by TAG).
used to exemplify the approach to natural samples in general. Isotopic labeling is beneficial but not essential for CMP-NMR. Generally, labeling is necessary when less sensitive multidimensional spectra (for example, DARR in this study) are being obtained to abbreviate spectrometer time with enhanced signalto-noise ratio. Natural abundance of 13C can be used to acquire NMR spectra but will increase sampling time considerably. The 13 C-labeled CP of wheat was performed over a period of 1 h and 43 min for 2K scans (signal-to-noise = 690), whereas the unlabeled equivalent containing 13C at the natural abundance took 16 h and 23 min for 20K scans (signal-to-noise = 69). Comparison of the 13C-labeled spectra to natural abundance spectra are provided in the Supporting Information (Figures S3−S5). In the future, the use of 7 mm rotor diameters will allow ∼4−5 times the sample volume to be introduced, which should substantially increase signal from unlabeled samples and should make similar studies on unlabeled material more feasible. The main drawback of CMP-NMR probes in
comparison to a dedicated HR-MAS or solid probe is loss of sensitivity. As discussed by Courtier Murias et al.,18 the fourchannel prototype probe used here suffers from a loss of ∼40% when compared to a two-channel solid probe. This loss mainly arises due to single circuit being quadrupley tuned in the fourchannel design. If identical probes could be compared (note, no two-channel CMP-NMR probes have been built to date), it is predicted that the loss in sensitivity would be ∼10−15% (mainly associated with the addition of the gradient coil). Due to the sensitivity losses CMP-NMR probes should be restricted to studies of native sample where the study of the components in their native phase is critical. In such cases the potential for CMP-NMR to provide otherwise inaccessible molecular-level information in situ is considerable. In studies following developmental changes it may be important to study the development of the solid, gel, and liquid components over time. Here it may be important to interleave solid, gel, and liquid NMR experiments and measure not just the phases independently but kinetic transfer between the phases. Such 113
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Funding
studies would be impossible using two or three separate NMR probes as it takes several hours to change and properly calibrate an NMR probe. Similarly, studies that follow the conversion of a molecule from liquid, to gel, to solid phases (for example, feeding phenylalanine to follow lignin growth or the binding and sequestration of a contaminant) also require the kinetic transfer between phases to be monitored and may find CMPNMR probes useful in the future. These examples indicate a future potential of CMP-NMR in seed/plant/food research. Finally, as the CMP-NMR probes combine all aspects of solution, HR-MAS, and solids, they provide the potential to develop novel NMR experiments (for example, solids using gradients) to better select and study structure and interaction in situ. Finally, it is important to note that quantification in complex systems such as seeds is complicated by spectral overlap. Spectral editing can extract molecules in the liquid, gel, semisolid, or solid state, and theoretically if the carbons were detected in exactly the same manner it would be possible to quantify the distribution between the different phases. However, in this study, this has been complicated by the fact CP-MAS was used for solid components, which enhances certain signals more than others, making this comparison inaccurate. Commonly, for quantification in solids, direct polarization magic angle spinning (DP-MAS)45 would be used. With regard to CMP-NMR, the cross-polarization element was required to select the rigid bonds (i.e., spectral editing to select the solid components), further complicating the issue. It may possible to quantify in the future, but this would require advanced techniques such as spin counting.46 Although the focus of this study has been to demonstrate the general applicability of CMP-NMR to the chemical structures of seeds, nonlabeled broccoli seeds were collected after spinning to test if they would germinate. They successfully germinated after being transferred to a Petri dish with a filter paper moist with water. This indicates that the seeds were still alive during spinning, and the potential for monitoring them while germinating the seed exists. CMP-NMR holds potential to provide an unprecedented window into the germination process itself. This avenue opens potential for research in fields such as physiology (germination, vernalization processes), agriculture (seed viability), and food safety (pathogen test, purity). Specifically, applications are possible in selecting viable seeds (current methods are mutagenic, destructive, or timeconsuming) or for seed selection breeding programs, which all require detailed yet nondestructive molecular analysis.47
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We thank the Natural Sciences and Engineering Research Council of Canada NSERC, Strategic and Discovery Grants Programs, the Canada Foundation for Innovation (CFI), the Ontario Ministry of Research and Innovation (MRI), and the Krembil Foundation for providing funding. A.J.S. thanks the Government of Ontario for an Early Researcher Award. We acknowledge the Dutch Innovation Program Food and Nutrition Delta for financial support (Project FND09002). Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank E. Koning and R. J. P. Baan (Koppert Cress BV) for kindly providing nonlabeled seeds of Brassica oleracea L. from which the 13C-labeled plants and seeds were produced. L.L. thanks A. J. Veloso for constructive criticism and editing of the manuscript.
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ASSOCIATED CONTENT
S Supporting Information *
Example COSY and TOCSY spectra with TAG assignments labeled; expansion of Figure 1c (HSQC of a single 13C labeled wheat seed) with brief description of the metabolites identified and figures comparing 13C-labeled spectra to natural abundance spectra of a single wheat seed. This material is available free of charge via the Internet at http://pubs.acs.org.
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