Structural Characterization of Monosialo-, Disialo- and Trisialo-gangliosides by Negative Ion AP-MALDI-QIT-TOF Mass Spectrometry with MS<Superscript>n</Superscript> Switching

The atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) is a quite convenient soft ionization for biomolecules, keeping analytes atmospheric conditions instead of high vacuum conditions. In this study, an AP-MALDI ion source has been coupled to a quadrupole ion trap time-of-flight (QIT-TOF) mass spectrometer, which is able to perform MSⁿ analysis. We applied this system to the structural characterization of monosialogangliosides, GM1 (NeuAc) and GM2 (NeuAc), disialogangliosides, GD2 (NeuAc, NeuAc), GD1a (NeuAc, NeuAc) and GD1b (NeuAc, NeuAc) and trisialoganglioside GT1a (NeuAc, NeuAc, NeuAc). In this system, the negative ion mass spectra of MS, MS² and MS³, a set of three mass spectra, were able to measure within 2 s per cycle. Thus, obtained results demonstrate that the negative ion mode MS, MS² and MS³ spectra provided sufficient information for the determination of molecular weights, oligosaccharide sequences and ceramide structures, and indicate that the AP-MALDI-QIT-TOF mass spectrometry keeping analytes atmospheric conditions with MSⁿ switching is quite useful and convenient for structural analyses of various types of sialic acid-containing GSLs, gangliosides.     

Structural characterization of neutral glycosphingolipids using high-performance liquid chromatography-electrospray ionization mass spectrometry with a repeated high-speed polarity and MS<Superscript>n</Superscript> switching system

Four types of neutral glycosphingolipids (LacCer, Gb₃Cer, Gb₄Cer, and IV³αGalNAc-Gb₄Cer; 10 pmol each) were analyzed using high-performance liquid chromatography (HPLC)-electrospray ionization quadrupole ion trap time-of-flight (ESI-QIT-TOF) mass spectrometry (MS) with a repeated high-speed polarity and MSⁿ switching system. This system can provide six types of mass spectra, including positive and negative ion MS, MS², and MS³ spectra, within 1 s per cycle. Using HPLC with a normal-phase column, information on the molecular weights of major molecular species of four neutral glycosphingolipids was obtained by detecting [M+Na]⁺ in the positive ion mode mass spectra and [M?H]^? in the negative ion mode mass spectra. Sequences of glycosphingolipid oligosaccharide were obtained in the negative ion MS² spectra. In addition, information on the ceramide structures was clearly obtained in the negative ion MS³ mass spectra. GlcCer molecular species were analyzed by HPLC-ESI-QIT-TOF MS with a reversed-phase column using 1 pmole of GlcCer. The structures of the seven molecular species of GlcCer, namely, d18:1-C16:0, d18:1-C18:0, d18:1-C20:0, d18:1-C22:0, d18:1-C23:0, d18:1-C24:1, and d18:1-C24:0, were characterized using positive ion MS and negative ion MS, MS², and MS³. The established HPLC-ESI-QIT-TOF MS with MSⁿ switching and a normal phase column has been successfully applied to the structural characterization of LacCer and Gb₄Cer in a crude mixture prepared from human erythrocytes.     

NMR,HS‐SPME‐GC/MS,and HPLC/MSn Analyses of Phytoconstituents and Aroma Profile of Rosmarinus eriocalyx

In this work, a comprehensive study on the chemical constituents of the aerial parts of Rosmarinus eriocalyx (Lamiaceae), an aromatic shrub traditionally consumed as a food and herbal remedy in Algeria, is presented. The aroma profile was analysed by headspace solid phase microextraction (HS‐SPME) coupled with gas chromatography‐mass spectrometry (GC/MS), whereas the crude extract constituents were analyzed by ¹H‐NMR and by high performance liquid chromatography coupled with mass spectrometry (HPLC/MSⁿ). Thirty‐nine volatile compounds, most of them being monoterpenes, have been identified, with camphor, camphene, and α‐pinene as the most abundant constituents. ¹H‐NMR analysis revealed the presence of phenolic compounds and betulinic acid while HPLC/MSⁿ allowed the identification of glycosilated and aglyconic flavonoids as well as phenylpropanoid derivatives. Some of these constituents, namely as betulinic acid, rosmanol, and cirsimaritin were reported for the first time in R. eriocalyx.     

Rare triterpene glycoside of ginseng (ginsenoside malonyl-Rg<Subscript>1</Subscript>) detected in plant cell suspension culture of <Emphasis Type="Italic">Panax japonicus</Emphasis> var. <Emphasis Type="Italic">repens</Emphasis>

This paper reports for the first time about the detection and identification of ginsenoside malonyl-Rg₁ (the rare 20(S)-protopanaxatriol-type ginsenoside) in the biomass of plant cell suspension culture of Japanese ginseng (Panax japonicus C.A. Mey. var. repens). Ginsenosides were analyzed by means of high-performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI-MS) in positive-ion mode. Malonyl-Rg1 was identified as a result of interpretation of MS spectra obtained upon fragmentation of protonated molecular ion ([M + H]⁺) of this compound in an ionization source. Chromatographic analysis and MS spectra showed that the cells of P. japonicus var. repens cultivated in vitro contain several isomers of malonyl-Rg₁. Thus, we ascertained for the first time that, in addition to malonyl ginsenosides of 20(S)-protopanaxadiol group, the plant cell culture of ginseng P. japonicus var. repens can accumulate glycosides of 20(S)-protopanaxatriol group acylated with a malonic acid residue. The obtained results showed that, in the cells of ginseng cultivated in vitro for a long time (for 10 years and more), the assortment of secondary metabolites (ginsenosides) may be as wide as in intact plants.     

Stronger anti-obesity effect of white ginseng over red ginseng and the potential mechanisms involving chemically structural/compositional specificity to gut microbiota

BackgroundGinseng has therapeutic potential for treating obesity and the associated gut microbiota dysbiosis. However, whether white ginseng and red ginseng, the two kinds of commonly used processed ginseng, possess different anti-obesity effects remains unknown.PurposeAnti-obesity effects of water extracts of white ginseng and red ginseng (WEWG and WERG) were compared, and the potential mechanisms were discussed.MethodsChemical profiles of WEWG and WERG were characterized by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry (UHPLC-QqQ-MS/MS) and high performance liquid chromatography coupled with evaporative light scattering detector (HPLC-ELSD). Anti-obesity effects of WEWG/WERG were examined by determining fat accumulation, systemic inflammation, enteric metabolic disorders and gut microbiota dysbiosis in high-fat diet (HFD)-fed obese mice.ResultsBoth WEWG and WERG exerted anti-obesity effects, with WEWG stronger than WERG. Compared to WERG, WEWG contained less contents of carbohydrates (polysaccharides, oligosaccharides, free monosaccharides) and ginsenosides, but chemical structures or compositions of these components in WEWG were characteristic, i.e. narrower molecular weight distribution and higher molar ratios of glucose residues of polysaccharides; higher content ratios of oligosaccharides DP2–3 (di-/tri-saccharides)-to-oligosaccharides DP4–7 (tetra-/penta-/hexa-/hepta-saccharides), sucrose-to-melibiose, maltose-to-trehalose and high-polar-to-low-polar ginsenosides. WEWG better ameliorated fat accumulation, enteric metabolic disorders and gut microbiota dysbiosis in HFD-fed obese mice than WERG.ConclusionThe stronger anti-obesity effect of white ginseng appears to correlate with differences in its chemical profile as compared to red ginseng. The carbohydrates and ginsenosides in WEWG potentially present more structural and compositional specificity to the obesity-associated gut bacteria, allowing more beneficial effects of WEWG on the gut microbiota dysbiosis. This consequently better alleviates the enteric metabolic disorders and systemic inflammation, thereby contributing to the stronger anti-obesity effect of WEWG as compared to WERG.     

Changes in gene expression during hairy root formation by<Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> infection in ginseng

Researchers have widely adopted the hairy root culture system as a means for producing secondary metabolites, including ginsenosides from ginseng. Although bacterial genes are involved, the aspects of plant gene expression are unclear. Using a cDNA microarray approach, we identified genes that are differentially expressed in ginseng hairy roots afterAgrobacterium rhizogenes infection. Our goal was to gain an initial understanding of the correlation between hairy root morphology and ginsenoside production. Among the 250 genes analyzed here, 63 (including 14 that are unclassified) were differentially expressed in a hairy root line containing a high level of ginsenosides. Of the genes that had been functionally categorized, 29% and 17% were active in metabolism and stress responses, respectively. Most were primarily associated with ribosomal proteins, thereby functioning in protein synthesis and destination. Their expression was down-regulated in hairy roots having less lateral branching. This phenotype may have resulted from the manipulation of metabolic activities by the translational machinery.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C, <Superscript>15</Superscript>N backbone assignment of the human heat-labile enterotoxin B-pentamer and chemical shift mapping of neolactotetraose binding

The major virulence factor of enterotoxigenic Escherichia coli is the heat-labile enterotoxin (LT), an AB₅ toxin closely related to the cholera toxin. LT consists of six subunits, the catalytically active A-subunit and five B-subunits arranged as a pentameric ring (LTB), which enable the toxin to bind to the epithelial cells in the intestinal lumen. LTB has two recognized binding sites; the primary binding site is responsible for anchoring the toxin to its main receptor, the GM₁-ganglioside, while the secondary binding site recognizes blood group antigens. Herein, we report the ¹H, ¹³C, ¹⁵N main chain assignment of LTB from human isolates (hLTB; 103 a.a. per subunit, with a total molecular mass of 58.5 kDa). The secondary structure was predicted based on ¹³C′, ¹³C^{α, 13}C^{β, 1}H^N and ¹⁵N chemical shifts and compared to a published crystal structure of LTB. Neolactotetraose (NEO) was titrated to hLTB and chemical shift perturbations were measured. The chemical shift perturbations were mapped onto the crystal structure, confirming that NEO binds to the primary binding site of hLTB and competes with GM₁-binding. Our new data further lend support to the hypothesis that binding at the primary binding site is transmitted to the secondary binding site of the toxin, where it may influence the binding to blood group antigens.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N backbone NMR chemical shift assignments of the C-terminal P4 domain of Ahnak

Ahnak is a ~?700 kDa polypeptide that was originally identified as a tumour-related nuclear phosphoprotein, but later recognized to play a variety of diverse physiological roles related to cell architecture and migration. A critical function of Ahnak is modulation of Ca²⁺ signaling in cardiomyocytes by interacting with the β subunit of the L-type Ca²⁺ channel (Ca_V1.2). Previous studies have identified the C-terminal region of Ahnak, designated as P3 and P4 domains, as a key mediator of its functional activity. We report here the nearly complete ¹H, ¹³C and ¹⁵N backbone NMR chemical shift assignments of the 11 kDa C-terminal P4 domain of Ahnak. This study lays the foundations for future investigations of functional dynamics, structure determination and interaction site mapping of the Ca_V1.2-Ahnak complex.     

Application of atmospheric pressure chemical ionisation mass spectrometry in the identification and differentiation of Panax species

An HPLC-MS method using an atmospheric pressure chemical ionisation (APCI) source has been developed to assist in the differentiation of three ginseng species: Panax quinquefolium (American ginseng), P. ginseng (Chinese ginseng) and P. notoginseng (sanqi) species. The differentiation method relies on the identification of ginsenosides Rf and F11 and notoginsenoside R1. R1 is observed in both P. notoginseng and Chinese ginseng, whilst F1, is found exclusively in the American species. The presence of these compounds permits the definitive identification of the species to be made. The APCI ionisation source has been employed to tackle the matrix interference in analysing Chinese medicinal materials and to minimise the associated matrix effects that are commonly encountered with other ionisation modes. Moreover, the method allows direct interface to conventional HPLC systems. More importantly, chemical reference standards of ginsenosides are not required in this method. This technique provides an alternative approach to analysing high molecular weight polar compounds that typically encountered in complex matrices of Chinese medicinal materials.     

Dimension-Enhanced Ultra-High Performance Liquid Chromatography/Ion Mobility-Quadrupole Time-of-Flight Mass Spectrometry Combined with Intelligent Peak Annotation for the Rapid Characterization of the Multiple Components from Seeds of <i>Descurainia sophia</i>

The complex composition of herbal metabolites necessitates the development of powerful analytical techniques aimed to identify the bioactive components. The seeds of Descurainia sophia (SDS) are utilized in China as a cough and asthma relieving agent. Herein, a dimension-enhanced integral approach, by combining ultra-high performance liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (UHPLC/IM-QTOF-MS) and intelligent peak annotation, was developed to rapidly characterize the multicomponents from SDS. Good chromatographic separation was achieved within 38 min on a UPLC CSH C18 (2.1 × 100 mm, 1.7 μm) column which was eluted by 0.1% formic acid in water (water phase) and acetonitrile (organic phase). Collision-induced dissociation-MS² data were acquired by the data-independent high-definition MS^E (HDMS^E) in both the negative and positive electrospray ionization modes. A major components knockout strategy was applied to improve the characterization of those minor ingredients by enhancing the injection volume. Moreover, a self-built chemistry library was established, which could be matched by the UNIFI software enabling automatic peak annotation of the obtained HDMS^E data. As a result of applying the intelligent peak annotation workflows and further confirmation process, a total of 53 compounds were identified or tentatively characterized from the SDS, including 29 flavonoids, one uridine derivative, four glucosides, one lignin, one phenolic compound, and 17 others. Notably, four-dimensional information related to the structure (e.g., retention time, collision cross section, MS¹ and MS² data) was obtained for each component by the developed integral approach, and the results would greatly benefit the quality control of SDS.     

Different chilling stresses stimulated the accumulation of different types of ginsenosides in <Emphasis Type="Italic">Panax ginseng</Emphasis> cells

Ginseng (Panax ginseng) is one of the most medically important plants in the world. Dammarane-type ginsenosides, which mainly include protopanaxatriol-type (PPT-type) and protopanaxadiol-type (PPD-type) ginsenosides, are the major pharmacologically relevant compounds that are produced by ginseng. Dammarenediol-II synthase (DDS) is the first committed enzyme in the ginsenoside biosynthetic pathway for dammarane-type ginsenosides, and PPD-type and PPT-type ginsenosides are catalyzed by protopanaxadiol synthase (PPDS) and protopanaxatriol synthase (PPTS), respectively. Ginseng cells are often used in stress studies. During their growth and development, ginseng plants are often exposed to cold stress. This study evaluated the effects of different chilling stresses on the accumulation of ginsenosides and the expressions of the DDS, PPDS and PPTS genes in ginseng cells. The results showed that continuous chilling (5 °C for 12 h) induced the PPT-type ginsenosides; whereas intermittent chilling (25 °C for 12 h and 5 °C for 12 h) stimulated the accumulation of PPD-type ginsenosides. The expression levels of DDS, PPDS and PPTS were clearly consistent with the accumulation pattern for PPT-type ginsenosides under continuous chilling stress or PPD-type ginsenosides under intermittent chilling stress, as was their order of involvement in the PPT-type or PPD-type biosynthetic pathway. These results indicate that different chilling treatments stimulated the accumulation of different types of ginsenosides, suggesting that cold stress may be one of the reasons for ginsenoside accumulation in ginseng cells.     

A simple method to adjust inconsistently referenced <Superscript>13</Superscript>C and <Superscript>15</Superscript>N chemical shift assignments of proteins

Inconsistent ¹³C and ¹⁵N chemical shift referencing is a continuing problem associated with protein chemical shift assignments deposited in BioMagResBank (BMRB). Here we describe a simple and robust approach that can quantitatively determine the ¹³C and ¹⁵N referencing offsets solely from chemical shift assignment data and independently of 3D coordinate data. This novel structure-independent approach permitted the assessment and determination of ¹³C and ¹⁵N reference offsets for all protein entries deposited in the BMRB. Tests on 452 proteins with known 3D structures show that this structure-independent approach yields ¹³C and ¹⁵N referencing offsets that exhibit excellent agreement with those calculated on the basis of 3D structures. Furthermore, this protocol appears to improve the accuracy of chemical shift-derived secondary structural identification, and has been formally incorporated into a computer program called PSSI (http//www.pronmr.com).Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-004-7441-3     

Analysis of novel over‐ and under‐sulfated glycosaminoglycan sequences by enzyme cleavage and multiple stage MS

We report on a novel strategy for identification of specific sulfation motifs in chondroitin/dermatan sulfate (CS/DS) chain derived from decorin (Dcn), based on enzyme cleavage and multistage MS (MSⁿ). Released CS/DS chains were digested with chondroitin B and in parallel with AC I lyases to obtain oligosaccharides of known hexuronic acid (HexA) epimerization. The depolymerized chains were separated by gel filtration, and collected di‐ and hexasaccharides were analyzed by ESI MSⁿ. MS² on bisulfated 4,5‐Δ‐HexAGalNAc revealed an additional sulfate ester group at 4,5‐Δ‐HexA. MS² data provided evidence upon GlcA sulfation in Dcn due to the fact that 4,5‐Δ‐HexA derived from GlcA after chondroitin AC I lyase treatment. Hexasaccharide screening in the MS¹ mode indicated direct correlation between the sulfate distribution and HexA epimerization. MSⁿ performed on ions that, according to mass calculation, correspond to pentasulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂], trisulfated [4,5‐Δ‐HexAGalNAc(GlcAGalNAc)₂] with IdoA‐derived 4,5‐Δ‐HexA at the nonreducing end, tetrasulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] and monosulfated [4,5‐Δ‐HexAGalNAc(IdoAGalNAc)₂] with GlcA‐derived 4,5‐Δ‐HexA at the nonreducing end rendered fragmentation patterns confirming the presence of over‐, regular, and under‐sulfated regions as well as structural motifs having both types of HexA sulfated within Dcn CS/DS.     

Broadband <Superscript>15</Superscript>N–<Superscript>13</Superscript>C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies

An approach for generating efficient RN_n^{n_S, n_k} {\rm{RN}}_{n}^{\nu_{\rm{S}}, {\nu_{\rm{k}}}} symmetry-based dual channel RF pulse schemes for γ-encoded broadband ¹⁵N–¹³C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic “R” element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic “R” element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and ¹⁵N–¹³C chemical shift correlation experiments. Employing such ¹³C–¹⁵N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.     

Critical test of some computational methods for prediction of NMR <Superscript>1</Superscript>H and <Superscript>13</Superscript>C chemical shifts

Performance of 18 DFT functionals (B1B95, B3LYP, B3PW91, B97D, BHandHLYP, BMK, CAM-B3LYP, HSEh1PBE, M06-L, mPW1PW91, O3LYP, OLYP, OPBE, PBE1PBE, tHCTHhyb, TPSSh, wB97xD, VSXC) in combinations with six basis sets (cc-pVDZ, aug-cc-pVDZ, cc-pVTZ, aug-cc-pVTZ, IGLO-II, and IGLO-III) and three methods for calculating magnetic shieldings (GIAO, CSGT, IGAIM) was tested for predicting ¹H and ¹³C chemical shifts for 25 organic compounds, for altogether 86 H and 88 C atoms. Proton shifts varied between 1.03 ppm to 12.00 ppm and carbon shifts between 7.87 ppm to 209.28 ppm. It was found that the best method for calculating ¹³C shifts is PBE1PBE/aug-cc-pVDZ with CSGT or IGAIM approaches (mae?=?1.66 ppm), for ¹H the best results were obtained with HSEh1PBE, mPW1PW91, PBE1PBE, CAM-B3LYP, and B3PW91 functionals with cc-pVTZ basis set and with CSGT or IGAIM approaches (mae?=?0.28 ppm). We found that often larger basis sets do not give better results for chemical shifts. The best basis sets for calculating ¹H and ¹³C chemical shifts were cc-pVTZ and aug-cc-pVDZ, respectively. CSGT and IGAIM NMR approaches can perform really well and are in most cases better than popular GIAO approach.

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Graphical Abstract Mean absolute errors for ¹H and ¹³C chemical shifts and computational times of neutral toluene molecule with aug-cc-pVDZ basis set and CSGT approach

     

Production of saponins from <Emphasis Type="Italic">Panax ginseng</Emphasis> suspension and adventitious root cultures

Biomass growth and ginsenoside production in cell suspension and adventitious roots of Panax ginseng C.A. Meyer cultures cultivated both in Erlenmayer flasks and a 3 dm³ bioreactor were studied. The maximum content of ginsenosides was found in the suspension culture cultivated in the bioreactor (4.34 % dry mass), however the saponin content was limited to two major ginsenosides, Rb₁ and Rg₁. The production of ginsenosides in adventitious roots was lower (1.45 or 1.72 % dry mass), nevertheless, the full range of ginsenosides was detected.This work was supported by 521/02/P064, COST 843.10, ME671 and Z4 055 905 projects.     

Structure characterization and identification steroidal saponins from Ophiopogon japonicus Ker-Gawler (Liliaceae) by high-performance liquid chromatography with ion trap mass spectrometry

Introduction – Steroidal saponins are the main active constituents in Ophiopogon japonicus Ker‐Gawler (Liliaceae). However, because of their high polarity, non‐chromophores and low content in plants, steroidal saponins are difficult to be isolated from O. japonicus by conventional phytochemical methods. Objective – To develop a sensitive and rapid approach towards the structural analysis of steroidal saponins using HPLC/ESI‐MSⁿ. Methodology – The fragmentation behaviors of six known steroidal saponins in negative ESI‐MSⁿ were used to deduce their mass spectral fragmentation mechanisms. By using HPLC/ESI‐MSⁿ, the important structural information on aglycone types, sugar types and saccharide sequences can be obtained. Results – According to the HPLC retention behaviour, the molecular structural characteristics provided by multistage mass spectrometry spectra and the literature, a total of 8 steroidal saponins were tentatively identified or characterized in O. japonicus rapidly. Conclusion – This work has shown that HPLC‐ESI‐MSⁿ may be used as an effective and rapid method for the characterization and identification of steroidal saponins from O. japonicus. Copyright © 2010 John Wiley & Sons, Ltd.     

Empirical correlation between protein backbone <Superscript>15</Superscript>N and <Superscript>13</Superscript>C secondary chemical shifts and its application to nitrogen chemical shift re-referencing

The linear analysis of chemical shifts (LACS) has provided a robust method for identifying and correcting ¹³C chemical shift referencing problems in data from protein NMR spectroscopy. Unlike other approaches, LACS does not require prior knowledge of the three-dimensional structure or inference of the secondary structure of the protein. It also does not require extensive assignment of the NMR data. We report here a way of extending the LACS approach to ¹⁵N NMR data from proteins, so as to enable the detection and correction of inconsistencies in chemical shift referencing for this nucleus. The approach is based on our finding that the secondary ¹⁵N chemical shift of the backbone nitrogen atom of residue i is strongly correlated with the secondary chemical shift difference (experimental minus random coil) between the alpha and beta carbons of residue i − 1. Thus once alpha and beta ¹³C chemical shifts are available (their difference is referencing error-free), the ¹⁵N referencing can be validated, and an appropriate offset correction can be derived. This approach can be implemented prior to a structure determination and can be used to analyze potential referencing problems in database data not associated with three-dimensional structure. Application of the LACS algorithm to the current BMRB protein chemical shift database, revealed that nearly 35% of the BMRB entries have δ ¹⁵N values mis-referenced by over 0.7 ppm and over 25% of them have δ ¹H^N values mis-referenced by over 0.12 ppm. One implication of the findings reported here is that a backbone ¹⁵N chemical shift provides a better indicator of the conformation of the preceding residue than of the residue itself. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

4D prediction of protein <Superscript>1</Superscript>H chemical shifts

A 4D approach for protein ¹H chemical shift prediction was explored. The 4th dimension is the molecular flexibility, mapped using molecular dynamics simulations. The chemical shifts were predicted with a principal component model based on atom coordinates from a database of 40 protein structures. When compared to the corresponding non-dynamic (3D) model, the 4th dimension improved prediction by 6–7%. The prediction method achieved RMS errors of 0.29 and 0.50 ppm for Hα and HN shifts, respectively. However, for individual proteins the RMS errors were 0.17–0.34 and 0.34–0.65 ppm for the Hα and HN shifts, respectively. X-ray structures gave better predictions than the corresponding NMR structures, indicating that chemical shifts contain invaluable information about local structures. The ¹H chemical shift prediction tool 4DSPOT is available from .     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung