Triple resonance experiments for aligned sample solid-state NMR of (13)C and (15)N labeled proteins

Initial steps in the development of a suite of triple-resonance (1)H/(13)C/(15)N solid-state NMR experiments applicable to aligned samples of (13)C and (15)N labeled proteins are described. The experiments take advantage of the opportunities for (13)C detection without the need for homonuclear (13)C/(13)C decoupling presented by samples with two different patterns of isotopic labeling. In one type of sample, the proteins are approximately 20% randomly labeled with (13)C in all backbone and side chain carbon sites and approximately 100% uniformly (15)N labeled in all nitrogen sites; in the second type of sample, the peptides and proteins are (13)C labeled at only the alpha-carbon and (15)N labeled at the amide nitrogen of a few residues. The requirement for homonuclear (13)C/(13)C decoupling while detecting (13)C signals is avoided in the first case because of the low probability of any two (13)C nuclei being bonded to each other; in the second case, the labeled (13)C(alpha) sites are separated by at least three bonds in the polypeptide chain. The experiments enable the measurement of the (13)C chemical shift and (1)H-(13)C and (15)N-(13)C heteronuclear dipolar coupling frequencies associated with the (13)C(alpha) and (13)C' backbone sites, which provide orientation constraints complementary to those derived from the (15)N labeled amide backbone sites. (13)C/(13)C spin-exchange experiments identify proximate carbon sites. The ability to measure (13)C-(15)N dipolar coupling frequencies and correlate (13)C and (15)N resonances provides a mechanism for making backbone resonance assignments. Three-dimensional combinations of these experiments ensure that the resolution, assignment, and measurement of orientationally dependent frequencies can be extended to larger proteins. Moreover, measurements of the (13)C chemical shift and (1)H-(13)C heteronuclear dipolar coupling frequencies for nearly all side chain sites enable the complete three-dimensional structures of proteins to be determined with this approach.     

The first in vivo observation of (13)C-(15)N coupling in mammalian brain.

[5-(13)C,(15)N]Glutamine, with (1)J((13)C-(15)N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by (13)C MRS at 4.7 T. The brain [5-(13)C]glutamine peak consisted of the doublet from [5-(13)C,(15)N]glutamine and the center [5-(13)C,(14)N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-(13)C,(15)N]glutamine was monitored in vivo with a time resolution of 20-35 min. This [5-(13)C,(15)N]glutamine was formed by glial uptake of released neurotransmitter [5-(13)C]glutamate and its reaction with (15)NH(3) catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively (13)C-enriched by intravenous [2,5-(13)C]glucose infusion to (13)C-label whole-brain glutamate C5, followed by [(12)C]glucose infusion to chase (13)C from the small and rapidly turning-over glial glutamate pool, leaving (13)C mainly in the neurotransmitter [5-(13)C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-(13)C,(15)N]glutamine arises from a coupling between (13)C of neuronal origin and (15)N of glial origin. Measurement of the rate of brain [5-(13)C,(15)N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

Below-ground partitioning (14C) and isotopic fractionation (delta13C) of carbon recently assimilated by maize

Partitioning of carbon recently assimilated by maize between shoots, roots, exudates, and CO2 from root respiration depending on three different levels of nutrient supply (full nutrient solution (NS), 10 times diluted NS, or deionised water) was estimated by 14C pulse labelling. A 13C fractionation in these compartments was investigated in relation to the nutrient supply. With decreasing nutrient supply, 14C allocation to the shoots and to the roots decreased from 76 % to 69 % and increased from 8 % to 13 % of 14C recovery, respectively. Average percentage of 14C in exudates and root-respired CO2 was 0.5 % and 16 % of 14C recovery, respectively. The concentration of the NS was not crucial for the amount of recently assimilated C recovered in exudates and CO2, but for the amounts in shoots and roots. For all three nutrient levels, roots were enriched in 13C when compared with shoots and 13C fractionation increased with decreasing nutrient supply up to 0.7 per thousand. Further 13C discrimination by exudation led to more 13C in exudates when compared with the roots of full nutrient supply and less 13C in exudates when compared with the roots grown in diluted NS and in deionised water. There were only small differences of<1.0 per thousand in delta13C values between roots and CO2 from root respiration. A 13C fractionation of recently assimilated C occurred between roots and exudates but was negligible for the CO2 respired by roots.     

(1)H-Detected IPAP DEPT-INADEQUATE and IPAP RINEPT-INADEQUATE for the measurement of long-range carbon-carbon coupling constants

The sensitivity of cryoprobes, which are rapidly becoming available, have brought about the possibility of measurement of (13)C, (13)C coupling constants at the natural abundance of (13)C using tens rather than hundreds of milligrams of compounds. This relatively recent development lays the foundation for a more routine use of the (13)C, (13)C long-range coupling constants in the conformational analysis of molecules. We have designed novel (1)H-detected INADEQUATE experiments optimized for long-range (13)C, (13)C correlations and the measurement of long-range coupling constants. These experiments incorporate refocusing of (1)J(CH) coupling constants prior to the formation of DQ coherences and (1)H-decoupling during the long carbon-carbon evolution intervals. Such modifications significantly enhance their performance over (1)H-detected INADEQUATE experiments currently in use for mapping the one-bond (13)C, (13)C correlations. (1)H or (13)C polarization is used a starting point in long-range correlation (1)H-detected IPAP DEPT-INADEQUATE or RINEPT-INADEQUATE experiments. These correlation experiments were modified yielding in-phase (IP) or antiphase (AP) (13)C, (13)C doublets in F(1). Procedures were developed for their editing yielding accurate values of small (13)C, (13)C coupling constants. The methods are illustrated using mono- and disaccharide samples and compared with related (13)C-detected experiments by means of the measurement of interglycosidic (13)C, (13)C coupling constants of a disaccharide.     

Double-quantum filtered rotational-echo double resonance

The homonuclear scalar coupling of a directly bonded 13C-13C pair has been used to create a double-quantum filter (DQF) to remove the natural-abundance 13C background in 13C{15N} rotational-echo double-resonance (REDOR) experiments. The DQF scalar and REDOR dipolar evolution periods are coincident which is important for sensitivity in the event of weak 13C-15N dipolar coupling. Calculated and observed 13C{15N} DQF-REDOR dephasings were in agreement for a test sample of mixed recrystallized labeled alanines. Glycine metabolism in a single uniform-15N soybean leaf labeled for 6 min by 13CO2 was measured quantitatively by 13C{15N} DQF-REDOR with no background interferences.     

Towards an inhalative 13C breath test method

Customary 13CO2 breath tests--and also 15N urine tests--always start with an oral administration of a test substrate. The test person swallows a stable isotope labelled diagnostic agent. This technique has been used to study several pathophysiological changes in gastrointestinal organs. However, to study pathophysiological changes of the bronchial and lung epithelium, the inhalative administration of a stable isotope labelled agent appeared more suitable to us. [1-13C]Hexadecanol and [1-13C]glucose were chosen. Inhaled [1-13C]hexadecanol did not yield 13CO2 in the exhaled air, but [1-13C]glucose did. To study the practicability of the [1-13C]glucose method and the reproducibility of the results, 18 inhalation tests were performed with healthy subjects. In 6 self-tests, the optimum inhalative dose of [13C]glucose was determined to be 205 mg. Using the APS aerosol provocation system with the nebulizer 'Medic Aid' (Erich Jaeger Würzburg), a 25% aqueous solution was inhaled. Then, breath samples were collected at 15 min. intervals and analysed for 13CO2. 75-120 min after the end of inhalation a well-reproducible maximum delta13C value of 6%o over baseline (DOB) was detected for 12 healthy probands. Speculating that the pulmonary resorption of the [13C]glucose is the rate-limiting step of elimination, decompensations in the epithelium ought to be reflected in changed [1-13C]glucose resorption rates and changed 13CO2 output. Therefore, we speculate that the inhalation of suitable 13C-labelled substrates will pave the way for a new group of 13CO2 breath tests aiding investigations of specific pathophysiological changes in the pulmonary tract, such as inflammations of certain sections and decompensations of cell functions.     

Automated data processing of [1H-decoupled] 13C MR spectra acquired from human brain in vivo

In clinical 13C infusion studies, broadband excitation of 200 ppm of the human brain yields 13C MR spectra with a time resolution of 2-5 min and generates up to 2000 metabolite peaks over 2h. We describe a fast, automated, observer-independent technique for processing [1H-decoupled] 13C spectra. Quantified 13C spectroscopic signals, before and after the administration of [1-13C]glucose and/or [1-13C]acetate in human subjects are determined. Stepwise improvements of data processing are illustrated by examples of normal and pathological results. Variation in analysis of individual 13C resonances ranged between 2 and 14%. Using this method it is possible to reliably identify subtle metabolic effects of brain disease including Alzheimer's disease and epilepsy.     

Towards hyperpolarized (13)C-succinate imaging of brain cancer

We describe a novel (13)C enriched precursor molecule, sodium 1-(13)C acetylenedicarboxylate, which after hydrogenation by PASADENA (Parahydrogen and Synthesis Allows Dramatically Enhanced Nuclear Alignment) under controlled experimental conditions, becomes hyperpolarized (13)C sodium succinate. Fast in vivo 3D FIESTA MR imaging demonstrated that, following carotid arterial injection, the hyperpolarized (13)C-succinate appeared in the head and cerebral circulation of normal and tumor-bearing rats. At this time, no in vivo hyperpolarized signal has been localized to normal brain or brain tumor. On the other hand, ex vivo samples of brain harvested from rats bearing a 9L brain tumor, 1 h or more following in vivo carotid injection of hyperpolarized (13)C sodium succinate, contained significant concentrations of the injected substrate, (13)C sodium succinate, together with (13)C maleate and succinate metabolites 1-(13)C-glutamate, 5-(13)C-glutamate, 1-(13)C-glutamine and 5-(13)C-glutamine. The (13)C substrates and products were below the limits of NMR detection in ex vivo samples of normal brain consistent with an intact blood-brain barrier. These ex vivo results indicate that hyperpolarized (13)C sodium succinate may become a useful tool for rapid in vivo identification of brain tumors, providing novel biomarkers in (13)C MR spectral-spatial images.     

应用显微激光拉曼光谱测定CO_2气体碳同位素值δ~(13)C的定量方法研究

单个流体包裹体同位素在研究岩矿古流体成因、矿床、油气和大地构造演化动力学等多个领域具有十分重要的意义,激光拉曼光谱是一项可以分析单个流体包裹体同位素的有效方法。本文提出应用显微激光拉曼光谱法来计算CO_2气体碳同位素值δ~(13)C。利用自行设计的装置将~(12)CO_2和~(13)CO_2按比例分别与N2混合,对混合气体样品进行显微激光拉曼测试分析后确定~(12)CO_2和~(13)CO_2的拉曼参数,这为用激光拉曼分析碳同位素值δ~(13)C奠定了理论基础。通过对不同比例的~(12)CO_2/~(13)CO_2人工合成CO_2包裹体样品和胜利油田CO_2天然气藏样品进行激光拉曼光谱分析,发现CO_2气体碳同位素摩尔分数比N_(13)/N_(12)与拉曼参数之间存在数学关系式,由此建立了根据碳同位素计算公式δ~(13)C=[(C_(13)/C_(12))_(样品)/(C_(13)/C_(12))_(标准)-1]×1 000‰,用激光拉曼分析获得的CO_2气体有关激光拉曼参数来计算δ~(13)C值的方法。按照该方法,应用显微激光拉曼光谱对胜利油田CO_2天然气藏样品分析计算其δ~(13)C值为-5.318‰,与用质谱仪分析测出的δ~(13)C值(-5.6‰)比较,其相对误差较小(≈5%),可以初步建立起应用显微激光拉曼光谱测定CO_2气体碳同位素值δ~(13)C的定量方法。     

Stable carbon and nitrogen isotope signatures of root-holoparasitic Cynomorium songaricum and its hosts at the Tibetan plateau and the surrounding Gobi desert in China

We first measured the δ(13)C and δ(15)N values of root holoparasite Cynomorium songaricum and its hosts from 19 sites across four provinces in northwest China, in an attempt to investigate their nutritional relationship at the Tibetan plateau and the surrounding Gobi desert. Our study showed that the δ(13)C of C. songaricum closely mirrored the values of its hosts, Nitraria tangutorum and N. sibirica across all sampling sites. C. songaricum was significantly depleted in (13)C compared to host plants at the Tibetan plateau, showing an average parasite/host δ(13)C difference of-0.6?‰. In contrast, (15)N of C. songaricum was significantly enriched by+1.3?‰ compared to the hosts, implying that these holoparasites had other nitrogen resources. Although no difference in the δ(13)C and δ(15)N values between holoparasites and hosts was detected, the δ(13)C and δ(15)N values of holoparasites were significantly correlated with those of their hosts at the Gobi desert. The δ(13)C versus δ(15)N values were significantly but negatively correlated for the hosts; however, holoparasite/host variation in δ(13)C was not correlated with the variation in δ(15)N. The δ(13)C versus δ(15)N values were negatively correlated in C. songaricum, and this relationship tended to be magnified along the increasing elevations independent of the host plants. C. songaricum at the Tibetan plateau exhibited different δ(13)C and δ(15)N signatures compared with those at the Gobi desert. Furthermore, both δ(13)C and δ(15)N values of C. songaricum and its host plants in salt marshes at the Tibetan plateau were different from those in sand sites at the Tibetan plateau and the Gobi desert. Our results indicate that the isotopic difference depends on the different altitudes and habitats and is host-specific.     

Frequency-selective heteronuclear dephasing and selective carbonyl labeling to deconvolute crowded spectra of membrane proteins by magic angle spinning NMR

We present a new method that combines carbonyl-selective labeling with frequency-selective heteronuclear recoupling to resolve the spectral overlap of magic angle spinning (MAS) NMR spectra of membrane proteins in fluid lipid membranes with broad lines and high redundancy in the primary sequence. We implemented this approach in both heteronuclear (15)N-(13)C(α) and homonuclear (13)C-(13)C dipolar assisted rotational resonance (DARR) correlation experiments. We demonstrate its efficacy for the membrane protein phospholamban reconstituted in fluid PC/PE/PA lipid bilayers. The main advantage of this method is to discriminate overlapped (13)C(α) resonances by strategically labeling the preceding residue. This method is highly complementary to (13)C(i-1)(')-(15)N(i)-(13)C(i)(α) and (13)C(i-1)(α)-(15)N(i-1)-(13)C(i)(') experiments to distinguish inter-residue spin systems at a minimal cost to signal-to-noise.     

Quantification of dung carbon incorporation in a temperate grassland soil following spring application using bulk stable carbon isotope determinations

Herbivore dung constitutes a substantial input of C to temperate grassland soils, and its fate must be determined in order to fully understand nutrient cycling in this ecosystem. This experiment used changes in bulk delta13C values of the 0-1 cm and 1-5 cm soil horizons of a dung-treated temperate grassland soil to approximate percentage applied dung C incorporation over 372 days. Natural abundance 13C-labelled C4 dung (delta13C - 12.6%) and C3 dung (delta13C - 31.3% were produced in a monitored diet switch from ryegrass silage (delta13C - 30.1%) to maize silage (delta13C - 11.6%). The dung was applied to a C3 grassland (delta13C 0-1 cm - 29.9%, 1-5 cm - 30.6%), and dung remains and soil cores from beneath the treatments were sampled at intervals. delta13C values were used to estimate a maximum of 12% applied dung C incorporation in the top 5 cm of the soil after 112 days, which declined to around 8% at the end of the experiment. A significant increase in percentage applied dung C was observed in the top 1 cm of soil, compared with the 1-5 cm horizon, after a substantial rain event after 30 days. However, results of forage fibre analyses of the two dung types revealed significant differences in composition which may affect subsequent calculations of percentage dung incorporation based on bulk delta13C values.     

Isotopic ((13)C) fractionation during plant residue decomposition and its implications for soil organic matter studies

Carbon isotopic fractionations in plant materials and those occurring during decomposition have direct implications in studies of short-and longer-term soil organic matter dynamics. Thus the products of decomposition, the evolved CO(2) and the newly formed soil organic matter, may vary in their (13)C signature from that of the original plant material. To evaluate the importance of such fractionation processes, the variations in (13)C signatures between and within plant parts of a tropical grass (Brachiaria humidicola) and tropical legume (Desmodium ovalifolium) were measured and the changes in (13)C content (signatures) during decomposition were monitored over a period of four months. As expected the grass materials were less depleted in (13)C (-11.4 to -11.9 per thousand) than those of the legume (-27.3 to -25.8 per thousand). Root materials of the legume were less (1.5 per thousand) depleted in (13)C compared with the leaves. Plant lignin-C was strongly depleted in (13)C compared with the bulk material by up to 2.5 per thousand in the legume and up to 4.7 per thousand in the grass. Plant materials were subsequently incubated in a sand/nutrient-solution/microbial inoculum mixture. The respiration product CO(2) was trapped in NaOH and precipitated as CaCO(3), suitable for analysis using an automated C/N analyser coupled to an isotope ratio mass spectrometer. Significant depletion in (13)C of the evolved CO(2) was observed during the initial stages of decomposition probably as a result of microbial fractionation as it was not associated with the (13)C signatures of the measured more decomposable fractions (non-acid detergent fibre and cellulose). While the cumulative CO(2)-(13)C signatures of legume materials became slightly enriched with ongoing decomposition, the CO(2)-C of the grass materials remained depleted in (13)C. Associated isotopic fractionation correction factors for source identification of CO(2-)C varied with time and suggested errors of 2-19% in the estimation of the plant-derived C at 119 days of incubation in a soil of an intermediate (-20.0 per thousand) (13)C signature. Analysis of the residual material after 119 days of incubation showed little or no change in the (13)C signature partly due to the incomplete decomposition at the time of harvesting. Copyright 1999 John Wiley & Sons, Ltd.     

Proton decoupled 13C NMR imaging

Proton decoupled 13C images were obtained at 2.1 Tesla. 13C[1H] images showed an increase in sensitivity over nondecoupled 13C images because of the nuclear Overhauser effect and elimination of multiple lines from scalar 13C-1H spin-spin couplings. The improvement in S/N for 13C[1H] images was smaller than expected because of a significant decrease in decoupling efficiency when 13C spin echoes were acquired in a readout gradient. Images of 13C compounds that had a wide range of chemical shifts showed separated and/or overlapping images, which is consistent with chemical shift imaging artifacts seen in 1H images. This work examines the technical constraints of acquiring and the difficulties of interpreting 13C[1H] images.     

Observation of nitrogen-14, carbon-13 indirect spin-spin coupling in high-resolution 13C CP/MAS spectra of solids.

The 13C CP/MAS NMR spectrum of [(n-C3H7)4N][Cd(SCN)3], 1, indicates the presence of three non-equivalent thiocyanate ligands, in agreement with the results of a recent single-crystal X-ray diffraction study. Examination of the 13C MAS line shapes allows direct measurement of the indirect spin-spin coupling constants, 1J(14N, 13C) = 16 +/- 1 Hz and 2J(111/113Cd, 13C) = 75 +/- 5 Hz, for the unique N-bonded thiocyanate ligand. This is the first reported measurement of 1J(14N, 13C) and 2J(111/113Cd, 13C) in the solid state. Possible reasons for the failure to observe 1J(14N, 13C) values in previous high-resolution 13C CP/MAS NMR studies are summarized.     

Feasibility of using hyperpolarized [1-13C]lactate as a substrate for in vivo metabolic 13C MRSI studies

The development of dynamic nuclear polarization in solution has enabled in vivo 13C MR studies at high signal-to-noise ratio following injection of prepolarized 13C substrates. While prior studies have demonstrated the ability to observe metabolism following injection of hyperpolarized 13C pyruvate, the goal of this study was to develop and test a new hyperpolarized agent for investigating in vivo metabolism, [1-13C]lactate. A preparation for prepolarized 13C lactate and the requisite dissolution media were developed to investigate the feasibility for in vivo 13C MRS/MRSI studies following injection of this hyperpolarized agent. This study demonstrated, for the first time, not only the ability to detect hyperpolarized [1-13C]lactate in vivo but also the metabolic products 13C pyruvate, 13C alanine and 13C bicarbonate following injection in normal rats. The use of 13C lactate as a substrate provided the opportunity to study the conversion of lactate to pyruvate in vivo and to detect the secondary conversions to alanine and bicarbonate through pyruvate. This study also demonstrated the potential value of this hyperpolarized agent to investigate in vivo lactate uptake and metabolism in preclinical animal models.     

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.     

The First in Vivo Observation of C–N Coupling in Mammalian Brain

[5-¹³C,¹⁵N]Glutamine, with ¹J(¹³C–¹⁵N) of 16 Hz, was observed in vivo in the brain of spontaneously breathing rats by ¹³C MRS at 4.7 T. The brain [5-¹³C]glutamine peak consisted of the doublet from [5-¹³C,¹⁵N]glutamine and the center [5-¹³C,¹⁴N]glutamine peak, resulting in an apparent triplet with a separation of 8 Hz. The time course of formation of brain [5-¹³C,¹⁵N]glutamine was monitored in vivo with a time resolution of 20–35 min. This [5-¹³C,¹⁵N]glutamine was formed by glial uptake of released neurotransmitter [5-¹³C]glutamate and its reaction with ¹⁵NH₃ catalyzed by the glia-specific glutamine synthetase. The neurotransmitter glutamate C5 was selectively¹³C-enriched by intravenous [2,5-¹³C]glucose infusion to ¹³C-label whole-brain glutamate C5, followed by [¹²C]glucose infusion to chase ¹³C from the small and rapidly turning-over glial glutamate pool, leaving ¹³C mainly in the neurotransmitter [5-¹³C]glutamate pool, which is sequestered in vesicles until release. Hence, the observed [5-¹³C,¹⁵N]glutamine arises from a coupling between ¹³C of neuronal origin and ¹⁵N of glial origin. Measurement of the rate of brain [5-¹³C,¹⁵N]glutamine formation provides a novel noninvasive method of studying the kinetics of neurotransmitter uptake into glia in vivo, a process that is crucial for protecting the brain from glutamate excitotoxicity.     

(13)C chemical shift and (13)C-(14)N dipolar coupling tensors determined by (13)C rotary resonance solid-state NMR

This work explores the utility of simple rotary resonance experiments for the determination of the magnitude and orientation of (13)C chemical shift tensors relative to one or more (13)C--(14)N internuclear axes from (13)C magic-angle-spinning NMR experiments. The experiment relies on simultaneous recoupling of the anisotropic (13)C chemical shift and (13)C--(14)N dipole--dipole coupling interactions using 2D rotary resonance NMR with RF irradiation on the (13)C spins only. The method is demonstrated by experiments and numerical simulations for the (13)C(alpha) spins in powder samples of L-alanine and glycine with (13)C in natural abundance. To investigate the potential of the experiment for determination of relative/absolute tensor orientations and backbone dihedral angles in peptides, the influence from long-range dipolar coupling to sequential (14)N spins in a peptide chain ((14)N(i)--(13)C(alpha)(i)--(14)N(i+1) and (14)N(i+1)--(13)C'(i)--(14)N(i) three-spin systems) as well as residual quadrupolar-dipolar coupling cross-terms is analyzed numerically.     

Obtaining molecular and structural information from 13C-14N systems with 13C FIREMAT experiments

The effect of dipolar coupling to 14N on 13C FIREMAT (five pi replicated magic angle turning) experiments is investigated. A method is developed for fitting the 13C FIREMAT FID employing the full theory to extract the 13C-14N dipolar and 13C chemical shift tensor information. The analysis requires prior knowledge of the electric field gradient (EFG) tensor at the 14N nucleus. In order to validate the method the analysis is done for the amino acids alpha-glycine, gamma-glycine, l-alanine, l-asparagine, and l-histidine on FIREMAT FIDs recorded at 13C frequencies of 50 and 100 MHz. The dipolar and chemical shift data obtained with this analysis are in very good agreement with the previous single-crystal 13C NMR results and neutron diffraction data on alpha-glycine, l-alanine, and l-asparagine. The values for gamma-glycine and l-histidine obtained with this new method are reported for the first time. The uncertainties in the EFG tensor on the resultant 13C chemical shift and dipolar tensor values are assessed.