Liquid chromatography–tandem quadrupole mass spectrometry and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry measurement of targeted metabolites of Methylobacterium extorquens AM1 grown on two different carbon sources

Complementary methods using liquid chromatography–tandem quadrupole mass spectrometry (LC–MS/MS) and comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC × GC–TOF-MS) were developed and applied to determine targeted metabolites involved in central carbon metabolism [including tricarboxylic acid cycle, serine cycle, ethylmalonyl-coenzyme A (ethylmalonyl-CoA) pathway and poly-β-hydroxybutyrate cycle] of the bacterium Methylobacterium extorquens AM1 grown on two carbon sources, ethylamine (C2) and succinate (C4). Nucleotides, acyl-CoAs and a few volatile metabolites in cell extracts of M. extorquens AM1 were readily separated using either hydrophilic interaction liquid chromatography or reversed-phase liquid chromatography, and detected with good sensitivity by MS/MS. However, volatile intermediates within a low mass range (<300 m/z), especially at low abundance (such as glyoxylic acid and others <500 nM), were more effectively analyzed by GC × GC–TOF-MS which often provided better sensitivity, resolution and reproducibility. The complementary nature of the LC-based and GC-based methods allowed the comparison of 39 metabolite concentrations (the lowest level was at 139.3 nM). The overlap between the LC-based and GC-based methods of seven metabolites provided a basis to check for consistency between the two methods, and thus provided some validation of the quantification accuracy. The abundance change of 20 intermediates further suggested differences in pathways linked to C2 and C4 metabolism.     

Handling within run retention time shifts in two-dimensional chromatography data using shift correction and modeling

The use of PARAFAC for modeling GC × GC-TOFMS peaks is well documented. This success is due to the trilinear structure of these data under ideal, or sufficiently close to ideal, chromatographic conditions. However, using temperature programming to cope with the general elution problem, deviations from trilinearity within a run are more likely to be seen for the following three cases: (1) compounds (i.e., analytes) severely broadened on the first column hence defined by many modulation periods, (2) analytes with a very high retention factor on the second column and likely wrapped around in that dimension, or (3) with fast temperature program rates. This deviation from trilinearity is seen as retention time-shifted peak profiles in subsequent modulation periods (first column fractions). In this report, a relaxed yet powerful version of PARAFAC, known as PARAFAC2 has been applied to handle this shift within the model step by allowing generation of individual peak profiles in subsequent first column fractions. An alternative approach was also studied, utilizing a standard retention time shift correction to restore the data trilinearity structure followed by PARAFAC. These two approaches are compared when identifying and quantifying a known analyte over a large concentration series where a certain shift is simulated in the successive first column fractions. Finally, the methods are applied to real chromatographic data showing severely shifted peak profiles. The pros and cons of the presented approaches are discussed in relation to the model parameters, the signal-to-noise ratio and the degree of shift.     

Comprehensive analysis of yeast metabolite GC x GC-TOFMS data: combining discovery-mode and deconvolution chemometric software

The first extensive study of yeast metabolite GC x GC-TOFMS data from cells grown under fermenting, R, and respiring, DR, conditions is reported. In this study, recently developed chemometric software for use with three-dimensional instrumentation data was implemented, using a statistically-based Fisher ratio method. The Fisher ratio method is fully automated and will rapidly reduce the data to pinpoint two-dimensional chromatographic peaks differentiating sample types while utilizing all the mass channels. The effect of lowering the Fisher ratio threshold on peak identification was studied. At the lowest threshold (just above the noise level), 73 metabolite peaks were identified, nearly three-fold greater than the number of previously reported metabolite peaks identified (26). In addition to the 73 identified metabolites, 81 unknown metabolites were also located. A Parallel Factor Analysis graphical user interface (PARAFAC GUI) was applied to selected mass channels to obtain a concentration ratio, for each metabolite under the two growth conditions. Of the 73 known metabolites identified by the Fisher ratio method, 54 were statistically changing to the 95% confidence limit between the DR and R conditions according to the rigorous Student's t-test. PARAFAC determined the concentration ratio and provided a fully-deconvoluted (i.e. mathematically resolved) mass spectrum for each of the metabolites. The combination of the Fisher ratio method with the PARAFAC GUI provides high-throughput software for discovery-based metabolomics research, and is novel for GC x GC-TOFMS data due to the use of the entire data set in the analysis (640 MB x 70 runs, double precision floating point).     

壳聚糖膜固定生物活性短肽RGDS的研究

为改善壳聚糖对细胞的特异性吸附,采用水溶性碳二亚胺将生物活性短肽精氨酸-甘氨酸-天冬氨酸-丝氨酸(RGDS)固定到壳聚糖膜的表面,采用X射线光电子能谱检测固定多肽前后的壳聚糖膜表面,发现反应后壳聚糖膜表面氮元素含量增大,Nls和Cls曲线拟合谱中酰胺键增多,表明RGDS短肽已固定到壳聚糖膜的表面;人角膜缘上皮细胞体外培养实验表明,固定RGDS后壳聚糖膜的细胞黏附率有了明显提高,固定RGDS后的壳聚糖膜在角膜组织工程支架等方面有更好的应用潜力。     

Astrophysical S-factors from asymptotic normalization coefficients

S-factors for direct capture reactions can be found at astrophysical energies from asymptotic normalization coefficients which provide the normalization of the tail of the overlap function. For example the overlap for ⁸B → ⁷Be+p defines the S-factor for ⁷Be (p, γ)⁸B. Peripheral transfer reactions offer a technique to determine these asymptotic normalization coefficients. As a test of the technique, the ¹⁶O(³He, d)¹⁷F reaction has been used to determine asymptotic normalization coefficients for transitions to the ground and first excited states of ¹⁷F. The S-factors for ¹⁶O(p, γ)¹⁷F calculated from these ¹⁷F → ¹⁶O+p asymptotic normalization coefficients are found to be in very good agreement with recent measurements. Following the same technique, the ¹⁰B(⁷Be, ⁸B)⁹Be and ¹⁴N(⁷Be, ⁸B)¹³C reactions have been used to measure the asymptotic normalization coefficient for ⁷Be(p, γ)⁸B. This result provides an indirect determination of S ₁₇(0).     

Increasing selectivity in comprehensive three-dimensional gas chromatography via an ionic liquid stationary phase column in one dimension

Recent advances in improving the selectivity and performance for a comprehensive, three-dimensional (3D) gas chromatograph (GC³) instrument are described. With GC³, two six-port diaphragm valves are utilized as the interfaces between three, in-series capillary columns housed in a standard GC instrument fitted with a high data acquisition rate flame ionization detector (FID). Modulation periods for sampling from one column to the next are set so that sufficient slices (i.e., modulations) are acquired by the subsequent dimension resulting in comprehensive data. We present GC³ instrumentation with significantly higher 3D peak capacity than previously reported. An average peak capacity production (i.e., per time) of 180 resolved peaks per minute was experimentally achieved for three representative analytes in a 3D diesel sample separation. This peak capacity production is about 4 times higher than our previous report. We also demonstrate the significant benefit of the added chemical selectivity of the three column GC³ instrument relative to a two column GC × GC instrument, in which one of the three columns is a triflate ionic liquid stationary phase column with a high selectivity for phosphonated compounds (i.e., di-methyl-methyl phosphonate, di-ethyl-methyl phosphonate and di-isopropyl-methyl phosphonate). Using all three separation dimensions, the 2D separation fingerprint of a diesel sample is simultaneously obtained along with selective information regarding the phosphonated compounds in the diesel samples in the additional dimension.     

Application of comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry method to identify potential biomarkers of perinatal asphyxia in a non-human primate model

Perinatal asphyxia is a leading cause of brain injury in infants, occurring in 2-4 per 1000 live births. The clinical response to asphyxia is variable and difficult to predict with current diagnostic tests. Reliable biomarkers are needed to help predict the timing and severity of asphyxia, as well as response to treatment. Two-dimensional gas chromatography-time-of-flight-mass spectrometry (GC×GC-TOFMS) was used herein, in conjunction with chemometric data analysis approaches for metabolomic analysis in order to identify significant metabolites affected by birth asphyxia. Blood was drawn before and after 15 or 18 min of cord occlusion in a Macaca nemestrina model of perinatal asphyxia. Postnatal samples were drawn at 5 min of age (n=20 subjects). Metabolomic profiles of asphyxiated animals were compared to four controls delivered at comparable gestational age. Fifty metabolites with the greatest change pre- to post-asphyxia were identified and quantified. The metabolic profile of post-asphyxia samples showed marked variability compared to the pre-asphyxia samples. Fifteen of the 50 metabolites showed significant elevation in response to asphyxia, ten of which remained significant upon comparison to the control animals. This metabolomic analysis confirmed lactate and creatinine as markers of asphyxia and discovered new metabolites including succinic acid and malate (intermediates in the Krebs cycle) and arachidonic acid (a brain fatty acid and inflammatory marker) as potential biomarkers. GC×GC-TOFMS coupled with chemometric data analysis are useful tools to identify acute biomarkers of brain injury. Further study is needed to correlate these metabolites with severity of disease, and response to treatment.     

Achieving high peak capacity production for gas chromatography and comprehensive two-dimensional gas chromatography by minimizing off-column peak broadening

By taking into consideration band broadening theory and using those results to select experimental conditions, and also by reducing the injection pulse width, peak capacity production (i.e., peak capacity per separation time) is substantially improved for one dimensional (1D-GC) and comprehensive two dimensional (GC×GC) gas chromatography. A theoretical framework for determining the optimal linear gas velocity (the linear gas velocity producing the minimum H), from experimental parameters provides an in-depth understanding of the potential for GC separations in the absence of extra-column band broadening. The extra-column band broadening is referred to herein as off-column band broadening since it is additional band broadening not due to the on-column separation processes. The theory provides the basis to experimentally evaluate and improve temperature programmed 1D-GC separations, but in order to do so with a commercial 1D-GC instrument platform, off-column band broadening from injection and detection needed to be significantly reduced. Specifically for injection, a resistively heated transfer line is coupled to a high-speed diaphragm valve to provide a suitable injection pulse width (referred to herein as modified injection). Additionally, flame ionization detection (FID) was modified to provide a data collection rate of 5kHz. The use of long, relatively narrow open tubular capillary columns and a 40°C/min programming rate were explored for 1D-GC, specifically a 40m, 180μm i.d. capillary column operated at or above the optimal average linear gas velocity. Injection using standard auto-injection with a 1:400 split resulted in an average peak width of ～1.5s, hence a peak capacity production of 40peaks/min. In contrast, use of modified injection produced ～500ms peak widths for 1D-GC, i.e., a peak capacity production of 120peaks/min (a 3-fold improvement over standard auto-injection). Implementation of modified injection resulted in retention time, peak width, peak height, and peak area average RSD%'s of 0.006, 0.8, 3.4, and 4.0%, respectively. Modified injection onto the first column of a GC×GC coupled with another high-speed valve injection onto the second column produced an instrument with high peak capacity production (500-800peaks/min), ～5-fold to 8-fold higher than typically reported for GC×GC.     

多谱线耦合系统辐射束缚问题的理论处理

提出一种处理多荧光谱线耦合系统辐射束缚问题的理论方法．这里的耦合包括激发态间的碰撞传能和当谱线重叠时对荧光的交叉吸收．首先将描述辐射束缚的Ｈｏｌｓｔｅｉｎ方程扩展为适用于描述多个激发能级且包括交叉耦合项的微分积分方程组，并将其纳入矢量、矩阵的形式．然后直接通过算符运算求解，给出按算符展开的级数解式．最后对级数解进行约化，导出一个只用有限项便可对系统行为进行完整描述的、远较简单截断近似优越的结果．还给出了一个双激发能级系统的计算实例