Development of RAST assay for determination of anti-Populus canadensis IgE antibodies

One of the frequent causes of pollen allergy in our region (Serbia, Yugoslavia) is the pollen of poplar (Populus canadensis). The aim of this study was to form RAST for the determination of specific anti-Populus canadensis IgE antibodies. Affinity purified and radiolabelled (I-125) MoAb E1 was used for forming assay for the determination of specific IgE. By titration of extract of poplarPopulus canadensis we determined that the quantity of 0.65 mL extract is needed for coupling of lg BrCN activated paper discs. Coupling was performed in Na₂CO3/NaHCO₃ buffer pH 10, on 4°C for 48h. Using this newely formed RAST, specific forPopulus canadensis, we have deteminated anti-Populus canadensis IgE antibodies as well as cross reactivity between pollens ofPopulus canadensis andPopulus deltoides.     

Property and Application of BACy‐Based Functional Hydrogels

Conventional polyacrylamide hydrogels prepared from the free radical polymerization between acrylamide and N,N′‐methylenebisacrylamide (NMBA) have been frequently used in the biochemical technique like the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to resolve protein mixtures. In this study, we have prepared an alternative polyacrylamide hydrogel from the cross‐linking of acrylamide and N,N′‐bisacrylylcystamine (BACy). In addition, we have compared the BACy‐based hydrogel with the NMBA‐based polyacrylamide hydrogel for their physical properties such as swelling ratio, shear modulus, crosslink density and morphology. Moreover, we further determined whether BACy‐based polyacrylamide hydrogel could be applied to SDS‐PAGE and proteomics research. The results showed that this type of hydrogel is capable of separating proteins and facilitates further in‐gel protein digestion and the following protein identifications by mass spectrometry. In summary, our study provides a basis for the putative application of BACy‐based hydrogels.     

The Infrared Multiphoton-Dissociation Spectra of Bromopropene Isomeric Cations

The infrared quasi-resonance stepwise-multiphoton-dissociation spectra of the molecular ions of 1-bromo-propene, 2-bromopropene, 3-bromopropene, and bromocyclopropane have been obtained at selected wavelengths between 9. 2 and 10. 7 μm in a Fourier -transform mass spectrometer. The resulting spectra and the photo fragmentation kinetics suggest that these molecular ions retain their identity upon ionization, and can, thus, provide fingerprint spectral identifications for the ions.     

The extent and effects of peptide sequence scrambling via formation of macrocyclic <Emphasis Type="Italic">b</Emphasis> ions in model proteins

The extent and effects of sequence scrambling in peptide ions during tandem mass spectrometry (MS/MS) have been examined using tryptic peptides from model proteins. Sequencescrambled b ions appeared in about 35% of 43 tryptic peptides examined under MS/MS conditions. In general, these ions had relatively low abundances with averages of 8% and 16%, depending on the instrumentation used. A few tryptic peptides gave abundant scrambled b ions in MS/MS. However, peptide and protein identifications under proteomic conditions with Mascot were not affected, even for these peptides wherein scrambling was prominent. From the 43 tryptic peptides that have been investigated, the conclusion is that sequence scrambling is unlikely to impact negatively on the accuracy of automated peptide and protein identifications in proteomics.     

MOLECULAR STRUCTURE OF A NOVEL POLYMORPHIC MODIFICATION OF PINOSTROBIN

5-Hydroxy-7-methoxyflavanone (pinostrobin) was isolated from buds of balsamic poplar (Populus balsamifieraL.). An x-ray structure analysis of its novel polymorphic modification is performed.     

Rapid differentiation among bacteria that cause gastroenteritis by use of low-resolution Raman spectroscopy and PLS discriminant analysis

Use of classical microbiological methods to differentiate bacteria that cause gastroenteritis is cumbersome but usually very efficient. The high cost of reagents and the time required for such identifications, approximately four days, could have serious consequences, however, mainly when the patients are children, the elderly, or adults with low resistance. The search for new methods enabling rapid and reagentless differentiation of these microorganisms is, therefore, extremely relevant. In this work the main microorganisms responsible for gastroenteritis, Escherichia coli, Salmonella choleraesuis, and Shigella flexneri, were studied. For each microorganism sixty different dispersions were prepared in physiological solution. The Raman spectra of these dispersions were recorded using a diode laser operating in the near infrared region. Partial least-squares (PLS) discriminant analysis was used to differentiate among the bacteria by use of their respective Raman spectra. This approach enabled correct classification of 100% of the bacteria evaluated and unknown samples from the clinical environment, in less time (∼10 h), by use of a low-cost, portable Raman spectrometer, which can be easily used in intensive care units and clinical environments.     

Characterization of Klason lignin samples isolated from beech and aspen using microbore column size‐exclusion chromatography

The present study is focused on the separation and characterization of lignin samples isolated by Klason method from European beech (Fagus sylvatica) broadleaf hardwood and European aspen (Populus tremula) broadleaf softwood by size‐exclusion chromatography. The separation was carried out using dimethylformamide as major component of the mobile phase and a 3 mm id microbore column packed with hydroxyethyl methacrylate gel, calibrated with polystyrene standards. The influence of mobile phase composition and sample solvent composition on the chromatographic behavior and molar mass distributions was investigated.     

Post-acquisition ETD spectral processing for increased peptide identifications

Tandem mass spectra (MS/MS) produced using electron transfer dissociation (ETD) differ from those derived from collision-activated dissociation (CAD) in several important ways. Foremost, the predominant fragment ion series are different: c- and z ^·-type ions are favored in ETD spectra while b- and y-type ions comprise the bulk of the fragments in CAD spectra. Additionally, ETD spectra possess charge-reduced precursors and unique neutral losses. Most database search algorithms were designed to analyze CAD spectra, and have only recently been adapted to accommodate c- and z ^·-type ions; therefore, inclusion of these additional spectral features can hinder identification, leading to lower confidence scores and decreased sensitivity. Because of this, it is important to pre-process spectral data before submission to a database search to remove those features that cause complications. Here, we demonstrate the effects of removing these features on the number of unique peptide identifications at a 1% false discovery rate (FDR) using the open mass spectrometry search algorithm (OMSSA). When analyzing two biologic replicates of a yeast protein extract in three total analyses, the number of unique identifications with a ∼1% FDR increased from 4611 to 5931 upon spectral pre-processing—an increase of ∼28. 6%. We outline the most effective pre-processing methods, and provide free software containing these algorithms.     

Chemical characterization of ozonated lignin solutions from corn (Zea mays) stalk and poplar (Populus deltoides) wood by capillary gas chromatography

Corn (Zea mays) stalk and poplar (Populus deltoides) wood lignin was converted into monomeric aromatic compounds and short chain aliphatic carboxylic acids. The main reaction products were separated and identified using capillary gas chromatography-mass spectrometry on an HP-5 column. The compounds were considered as positively identified when their mass spectra and GC retention times agreed with those of authentic standard samples. The quantitative estimation of the identified reaction products was accomplished on an OV-101 capillary column by gas chromatography-FID using the internal standard method. Among the aromatic compounds, aldehydes (p-hydroxybenzaldehyde, vanillin, and syringaldehyde), acids (p-hydroxybenzoic, vanillic, 3,4-dihydroxybenzoic, and syringic), and one phenol (hydroquinone) were determined. In addition, the aliphatic carboxylic acids: glycolic, oxalic, malonic, glyoxylic, butanedioic, glyceric, and malic acid were identified. All the calibration curves of the quantified compounds approximated to a straight line. For both corn stalk and poplar wood lignins, the major components were the aromatic aldehydes (71 and 64% of the characterized fraction, respectively), followed by the aliphatic carboxylic acids (20 and 21% of the characterized fraction, respectively).     

Determination of bioconcentration factors of isomeric polycyclic aromatic hydrocarbons in polychaete worms by HRGC on selective liquid crystalline stationary phases

The bioconcentration factors (K_b) of parent and alkylated PAHs isolated from polychaete worms collected in coastal sediments were determined for the first time. Capillary gas chromatography using a polymeric liquid crystalline (MPMS) and SE-54 stationary phases coupled to FID, MS and concurrent elution with authentic standards permitted positive identifications of parent PAHs. The K_b values of 18 compounds were accurately determined on the liquid crystalline column and the those of mono- and dialkyl-phenathrene, -pyrene and -chrysene were determined by GC-MS on a SE-54 coated column. The experimental K_b values exhibited a moderate correlation (r=0.7–0.8) with the reported octanol-water partition coefficients (K_ow) for these compounds. Consequently, kinetic considerations, i.e. biouptake-release and enzymatic activity seem to be relevant, in addition of biota-media equilibrium. Therefore, integrated bioconcentration models are needed.     

Assessment of resolution parameters for CID-based shotgun proteomic experiments on the LTQ-Orbitrap mass spectrometer

Shotgun proteomics has been used extensively for characterization of a number of proteomes. High-resolution Fourier transform mass spectrometry (FTMS) has emerged as a powerful tool owing to its high mass accuracy and resolving power. One of its major limitations, however, is that the confidence level of peptide identification and sensitivity cannot be maximized simultaneously. Although it is generally assumed that higher resolution is better for peptide identifications, the precise effect of varying resolution as a parameter on peptide identification has not yet been systematically evaluated. We used the Escherichia coli proteome and a standard 48 protein mix to study the effect of different resolution parameters on peptide identifications in the setting of a shotgun proteomics experiment on an LTQ-Orbitrap mass spectrometer. We observed a higher number of peptide-spectrum matches (PSMs) whenever the MS scan was carried out by FT and the MS/MS in the ion-trap (IT) with the maximum PSMs obtained at an MS resolution of 30,000. In contrast, when samples were analyzed by FT for both MS and MS/MS, the number of PSMs was significantly lower (∼40% compared with FT-IT experiments) with the maximum PSMs obtained when both the MS and MS/MS resolution were set to 15,000. Thus, a 15K-15K resolution setting may provide the best compromise for studies where both speed and accuracy such as high-throughput post-translational analysis and de novo sequencing are important. We hope that our study will allow researchers to choose between different resolution parameters to achieve their desired results from proteomic analyses.     

Black poplar-tree (<Emphasis Type="Italic">Populus nigra</Emphasis> L.) bark as an alternative indicator of urban air pollution by chemical elements

Capabilities of black poplar-tree (Populus nigra L.) bark as a biomonitor of atmospheric air pollution by chemical elements were tested against epiphytic lichens Xanthoria parietina (L.) and Physcia adscendens (Fr.). Concentrations of 40 macro and trace elements were determined using epicadmium and instrumental NAA. The data obtained were processed using non-parametric tests. A good correlation was found between concentrations of majority of elements in bark and lichens. On the accumulation capability bark turned out to be competitive with both lichens examined. The main inorganic components of black poplar-tree bark were revealed. A substrate influence on the concentrations of some elements in epiphytic lichens was established. An optimized procedure of bark pre-irradiation treatment was suggested.     

Software Lock Mass by Two-Dimensional Minimization of Peptide Mass Errors

Mass accuracy is a key parameter in proteomic experiments, improving specificity, and success rates of peptide identification. Advances in instrumentation now make it possible to routinely obtain high resolution data in proteomic experiments. To compensate for drifts in instrument calibration, a compound of known mass is often employed. This ‘lock mass’ provides an internal mass standard in every spectrum. Here we take advantage of the complexity of typical peptide mixtures in proteomics to eliminate the requirement for a physical lock mass. We find that mass scale drift is primarily a function of the m/z and the elution time dimensions. Using a subset of high confidence peptide identifications from a first pass database search, which effectively substitute for the lock mass, we set up a global mathematical minimization problem. We perform a simultaneous fit in two dimensions using a function whose parameterization is automatically adjusted to the complexity of the analyzed peptide mixture. Mass deviation of the high confidence peptides from their calculated values is then minimized globally as a function of both m/z value and elution time. The resulting recalibration function performs equal or better than adding a lock mass from laboratory air to LTQ-Orbitrap spectra. This ‘software lock mass’ drastically improves mass accuracy compared with mass measurement without lock mass (up to 10-fold), with none of the experimental cost of a physical lock mass, and it integrated into the freely available MaxQuant analysis pipeline ().     

应用非同质荷比规则提高蛋白质组学检索结果的可信度和灵敏度

如何筛选合理的数据库匹配结果对于基于质谱的蛋白质组学研究至关重要。但是目前,基于打分体系和反转数据库的筛选方法都无法有效的避免假阳性和假阴性匹配的存在。因此,本文提出了一种系统的搜索策略: 非同质荷比检索规则 (INMZS)。在该策略中,所有匹配结果都需要检查相关匹配质荷比的分享程度,只有那些相关质荷比均为专有匹配时,蛋白质才会被作为可信结果保留,策略还采用迭代搜索方法以提高鉴定低丰度组分的灵敏度。最终,所有的匹配结果由诱饵数据库方法进行评估以得到最终结果列表。INMZS策略在标准蛋白质混合物和大规模人肝蛋白质组数据上进行了模拟及应用,结果显示,INMZS规则和诱饵数据库评估方法的结和可以有效的保证蛋白质组学数据匹配的可信度及灵敏度,可以广泛适用于基于二维凝胶电泳及非shotgun技术的蛋白组学研究中。     

Identification of autoxidation oligomers of flavan‐3‐ols in model solutions by HPLC‐MS/MS

Autoxidation of flavan‐3‐ols was carried out in aqueous/methanol model solutions under mildly acidic conditions (pH 6.0), and these autoxidation products were analyzed by using high performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS). The results showed that (+)‐catechins and (?)‐epicatechins generated autoxidation reaction with each other to form a series of oligomers that had the same [M ? H]^? molecular ions (MS¹) as those of natural procyanidins, but had completely different fragment ions (MS²). According to MS/MS analysis, the major fragments of these oligomers were derived not only from the retro‐Diels–Alder (RDA) dissociations on the C‐rings of the flavan‐3‐ol units, but also from the quinone‐methide (QM) cleavage of the interflavan linkages (IFL), and thus they were identified as B‐type dehydrodicatechins, B‐type dehydrotricatechins and A‐type dehydrotricatechins, respectively. The potential structures of their [M ? H]^? molecular ions and partial fragment ions were deduced on the basis of the MS/MS characterization and the oxidation of flavan‐3‐ols in previous reports. Some specific fragment ions were found to be very useful for identifying the autoxidation oligomers (the B‐type dehydrodicatechins at m/z 393, the B‐type dehydrotricatechins at m/z 681 and the A‐type dehydrotricatechins at m/z 725). Copyright © 2008 John Wiley & Sons, Ltd.     

A Pair of New Nortriterpene Saponin Epimers from the Roots of Gypsophila oldhamiana

A pair of new oleanane‐type nortriterpene saponin epimers, neogypsoside A ( 1 ) and B ( 2) (Fig. 1) with neogypsogenin A ( 3 ) and neogypsogenin B ( 4 ) as the two new aglycons, as well as the two known triterpene saponins 5 and 6 (Fig. 1), were isolated from the roots of Gypsophila oldhamiana. Their structures were determined by analysis of their NMR data. A possible biogenetic pathway to the nortriterpene saponins 1 and 2 is proposed (Scheme 2).     

Identification of NTBC metabolites in urine from patients with hereditary tyrosinemia type 1 using two different mass spectrometric platforms: triple stage quadrupole and LTQ‐Orbitrap

The objective of our work was to identify known and unknown metabolites of the drug NTBC (2‐(2‐nitro‐4‐trifluoromethylbenzoyl)‐1,3‐cyclohexanedione) in urine from patients during the treatment of hereditary tyrosinemia type 1 (HT‐1) disease, a severe inborn error of tyrosine metabolism. Two different mass spectrometric techniques, a triple stage quadrupole and an LTQ‐Orbitrap (Fourier transform mass spectrometry (FTMS)), were used for the identification and the structural elucidation of the detected metabolites. Initially, the mass spectrometric (MS) approach consisted of the precursor ion scan detection of the selected product ions, followed by the corresponding collision‐induced dissociation (CID) fragmentation analysis (MS²) for the targeted selected reaction monitoring (SRM) mode. Subsequently, accurate and high‐resolution full scan and MS/MS measurements were performed on the possible metabolites using the LTQ‐Orbitrap. Final confirmation of the identified metabolites was achieved by measuring commercially supplied or laboratory‐synthesized standards. Altogether six metabolites, including NTBC itself, were extracted, detected and identified. In addition, two new NTBC metabolites were unambiguously identified as amino acid conjugates, namely glycine‐NTBC and β‐alanine‐NTBC. These identifications were based on their characteristics of chromatographic retention times, protonated molecular ions, elemental compositions, product ions (using CID and higher‐energy C‐trap dissociation (HCD) techniques) and synthesized references. The applied MS strategy, based on two different MS platforms (LC/MS/MS and FTMS), allowed the rapid identification analysis of the drug metabolites from human extracts and could be used for pharmaceutical research and drug development. Copyright © 2010 John Wiley & Sons, Ltd.     

Synthesis,structure and antiproliferative activity of dinuclear ruthenium arene complexes with differently coordinated thiosemicarbazones

A series of di‐nuclear ruthenium arene complexes with TSC ligands ([(η⁶‐p‐cymene)Ru(N¹,S‐TSC)]₂Cl₂, A‐type, 1 and 2 ) and their corresponding analogues ([(η⁶‐p‐cymene)Ru(N²,S‐TSC)]₂Cl₂, B‐type, 3 and 4 ), in which TSCs act as different coordination mode, have been synthesized and structurally characterized by a variety of physical methods. The molecular structures of 1 , 3 and 4 were determined using single‐crystal X‐ray diffraction analysis. The Gibbs free energy of the two examples of the two types of complexes ( 1 and 3 ) and bonding order in their single‐crystals were discussed using density functional theory (DFT) calculations. The compounds were further evaluated for their in vitro antiproliferative activities against several cancerous and HEK‐293 T noncancerous cell lines, and the results indicate that B‐type complexes show stronger cytotoxicity than A‐type complexes. Furthermore, the interactions of the compounds with DNA were investigated by electrophoretic mobility spectrometry studies.     

Isobenzopyryliumsalts: VII—13C n.m.r. shifts of 1-arylisobenzopyrylium salts and correlation with Hammett parameters

The signal identifications of isobenzopyrylium salts has been achieved on the basis of proton broad-band and off-resonance decoupled ¹³C n.m.r. spectra of twelve different 1-arylisobenzopyrylium salts and eight model compounds. From the ¹³C shifts valuable information about pK values, the aromatic character and the conjugative effects of isobenzopyrylium salts was obtained.     

Structure and Metabolic‐Flow Analysis of Molecular Complexity in a 13C‐Labeled Tree by 2D and 3D NMR

Improved signal identification for biological small molecules (BSMs) in a mixture was demonstrated by using multidimensional NMR on samples from ¹³C‐enriched Rhododendron japonicum (59.5 atom%) cultivated in air containing ¹³C‐labeled carbon dioxide for 14 weeks. The resonance assignment of 386 carbon atoms and 380 hydrogen atoms in the mixture was achieved. 42 BSMs, including eight that were unlisted in the spectral databases, were identified. Comparisons between the experimental values and the ¹³C chemical shift values calculated by density functional theory supported the identifications of unlisted BSMs. Tracing the ¹³C/¹²C ratio by multidimensional NMR spectra revealed faster and slower turnover ratios of BSMs involved in central metabolism and those categorized as secondary metabolites, respectively. The identification of BSMs and subsequent flow analysis provided insight into the metabolic systems of the plant.