13C标记代谢流分析中天然稳定性同位素修正矩阵构建方法及应用

稳定性同位素¹³C标记实验是分析细胞代谢流的一种重要手段,主要通过质谱检测胞内代谢物中¹³C标记的同位素分布,并作为胞内代谢流计算时的约束条件,进而通过代谢流分析算法得到相应代谢网络中的通量分布。然而在自然界中,并非只有C元素存在天然稳定性同位素¹³C,其他元素如O元素也有其天然稳定性同位素¹⁷O、¹⁸O等,这使得质谱方法所测得的同位素分布中会夹杂除¹³C标记之外的其他元素的同位素信息,特别是分子中含有较多其他元素的分子,这将导致很大的实验误差,因此需要在进行代谢流计算前进行质谱数据的矫正。本研究提出了一种基于Python语言的天然同位素修正矩阵的构建方法,用于修正同位素分布测量值中由于天然同位素分布引起的测定误差。文中提出的基本修正矩阵幂方法用于构建各元素修正矩阵,结构简单、易于编码实现,可直接应用于¹³C代谢流分析软件数据前处理。将该修正方法应用于¹³C标记的黑曲霉（Aspergillus niger）胞内代谢流分析,结果表明本研究提出的方法准确有效,为准确获取微生物胞内代谢流分析提供了可靠的数据修正方法。     

Rapid measurement of plasma free fatty acid concentration and isotopic enrichment using LC/MS

Measurements of plasma free fatty acids (FFA) concentration and isotopic enrichment are commonly used to evaluate FFA metabolism. Until now, gas chromatography-combustion-isotope ratio mass spectrometry (GC/C/IRMS) was the best method to measure isotopic enrichment in the methyl derivatives of ¹³C-labeled fatty acids. Although IRMS is excellent for analyzing enrichment, it requires time-consuming derivatization steps and is not optimal for measuring FFA concentrations. We developed a new, rapid, and reliable method for simultaneous quantification of ¹³C-labeled fatty acids in plasma using high-performance liquid chromatography-mass spectrometry (HPLC/MS). This method involves a very quick Dole extraction procedure and direct injection of the samples on the HPLC system. After chromatographic separation, the samples are directed to the mass spectrometer for electrospray ionization (ESI) and analysis in the negative mode using single ion monitoring. By employing equipment with two columns connected parallel to a mass spectrometer, we can double the throughput to the mass spectrometer, reducing the analysis time per sample to 5 min. Palmitate flux measured using this approach agreed well with the GC/C/IRMS method. This HPLC/MS method provides accurate and precise measures of FFA concentration and enrichment.     

Radiocarbon dating: The continuing revolution

Radiocarbon (¹⁴C) dating, now in its fifth decade of general use, continues to be the most widely employed method of inferring chronometric age for late Pleistocene and Holocene age materials recovered from archeological contexts. Over the last decade, several technical advances in ¹⁴C studies have provided contexts for a number of significant applications in archeology that were previously either not possible or not practical. These include the extension of the calibrated ¹⁴C time scale into the late Pleistocene and the development of accelerator mass spectrometry (AMS). The contribution of AMS-based ¹⁴C values to the critical evaluation of archeological data is illustrated by considering the problems of dating early plant domestication in the Near East and Mesoamerica, New World Paleoindian human skeletal materials, and European Upper Paleolithic and Mesolithic materials.     

Disclosure of new and recurrent microbial metabolites by mass spectrometric methods

The application of modern mass spectrometry methods (SI-CID-MS/MS; MS ⁿ ) in the disclosure of new and recurrent microbial metabolites is discussed. Spray ion (SI) sources coupled to different kinds of mass analyzers enable the determination of molecular weights and chemical formulas of given samples even in mixtures. Diagnostic fragment formation by collision-induced dissociation (CID-MS/MS) and MS ⁿ experiments using ion trap mass analyzers are shown as another indispensable source of structural information. Due to the development of benchtop-type mass spectrometers coupled to high-performance liquid chromatography (HPLC), MS can be practised in almost every laboratory as a powerful tool in natural product analysis. Examples are given for special MS applications in identification of bioactive metabolites from screening strains. Journal of Industrial Microbiology & Biotechnology (2001) 27, 136–143. Received 21 September 1999/ Accepted in revised form 19 January 2000     

Use of secondary ion mass spectrometry to image44calcium uptake in the cell walls of apple fruit

Summary Calcium, an important agent in regulating cell wall autolysis during fruit ripening, interacts with pectic acid polymers to form cross-bridges that influence cell separation. In the present study, secondary ion mass spectrometry (SIMS) was used to determine whether the cell walls of apple fruit were able to take up exogenously applied⁴⁴Ca, which was infiltrated into mature fruit. SIMS, which has the ability to discriminate between isotopes, allowed localization of the exogenously applied⁴⁴Ca and the native⁴⁰Ca. The results indicated that the total amount of calcium present in the cell walls was enriched with⁴⁴Ca and that heterogeneity of⁴⁴Ca distribution occurred in the pericarp. Isotope ratio images showed microdomains in the cell wall, particularly in the middle lamella intersects that oppose the intercellular spaces. These domains may be the key areas that control cell separation. These data suggest that exogenously applied calcium may influence cell wall autolysis.Abbreviations SIMS secondary ion mass spectrometry     

An improved scheme of leucine derivative fragmentation in mass spectrometry

Summary This study illustrates the contribution of stable isotopes to amino acid mass spectrometry. The mass spectra of natural leucine and 1¹³C leucine were compared and the mass fragmentography pattern analysed. This analysis indicates that the position of the stable isotope in tracer molecules should be very dependent on the analytical procedure used for their determination.     

Determination of leucine flux in vivo by gas chromatography—mass spectrometry utilizing stable isotopes for trace and internal standard

A simple and reliable method is described for the determination of leucine flux in vivo using two stable isotopes of leucine and gas chromatography—mass spectrometry (GC---MS). [6,6,6-²H₃]Leucine is administered as a primed-dose constant infusion in vivo and -[²H₇]leucine is added to plasma as an internal standard. Plasma leucine concentration and moles per cent enrichment of [²H₃]leucine can be determined simultaneously by GC---MS and selected ion monitoring. Leucine flux calculated from the [6,6,6-²H₃]leucine data was nearly identical to that obtained with -[U-¹⁴C]leucine in dogs. This method is readily applicable to the study of leucine metabolism in humans of all ages and laboratory animals.     

Zinc,copper, and iron nutrition studied with enriched stable isotopes

Enriched stable isotopes were used in nutrition studies of normal, healthy adults to measure zinc, copper, and iron absorption. After obtaining baseline values for zinc, copper, and iron absorption from diets adequate in all nutrients, the effects of age, pregnancy, and deveral dietary variables were studied. Stable isotopes of zinc, copper, and iron were incorporated into diets. Complete fecal samples were collected and the unabsorbed isotopes remaining in the samples were measured by thermal ionization mass spectrometry, the most precise analytical method for the determination of stable mineral isotopes. Stable isotopes were also infused in five young men to evaluate the potential of studying mineral utilization and kinetics with stable isotopes. The results of these studies demonstrate that a number of factors can affect mineral absorption, but the specific effects differ for different minerals. Isotopic enrichments could be measured in urine and blood, so kinetic studies of utilization of essential minerals are now feasible with enriched stable isotopes. Continued use of stable isotopes to determine mineral absorption, combined with stable-isotope studies of mineral utilization, balance data, and biochemical indicators of mineral status, should result in a better understanding of mineral requirements and metabolism under a variety of conditions.     

Allocation and residence time of photosynthetic products in a boreal forest using a low-level 14C pulse-chase labeling technique

Much of our understanding about how carbon (C) is allocated in plants comes from radiocarbon (¹⁴C) pulse‐chase labeling experiments. However, the large amounts of ¹⁴C required for decay‐counting mean that these studies have been restricted for the most part to mesocosm or controlled laboratory experiments. Using the enhanced sensitivity for ¹⁴C detection available with accelerator mass spectrometry (AMS), we tested the utility of a low‐level ¹⁴C pulse‐chase labeling technique for quantifying C allocation patterns and the contributions of different plant components to total ecosystem respiration in a black spruce forest stand in central Manitoba, Canada. All aspects of the field experiment used ¹⁴C at levels well below regulated health standards, without significantly altering atmospheric CO₂ concentrations. Over 30 days following the label application in late summer (August and September), we monitored the temporal and spatial allocation patterns of labeled photosynthetic products by measuring the amount and ¹⁴C content of CO₂ respired from different ecosystem components. The mean residence times (MRT) for labeled photosynthetic products to be respired in the understory (feather mosses), canopy (black spruce), and rhizosphere (black spruce roots and associated microbes) were <1, 6, and 15 days, respectively. Respiration from the canopy and understory showed significantly greater influence of labeled photosynthates than excised root and intact rhizosphere respiration. After 30 days,∼65% of the label assimilated had been respired by the canopy,∼20% by the rhizosphere, and∼9% by the understory, with∼6% unaccounted for and perhaps remaining in tissues. Maximum ¹⁴C values in root and rhizosphere respiration were reached 4 days after label application. The label was still detectable in root, rhizosphere and canopy respiration after 30 days; these levels of remaining label would not have been detectible had a ¹³C label been applied. Our results support previous studies indicating that a substantial portion of the C fueling rhizosphere respiration in the growing season may be derived from stored C pools rather than recent photosynthetic products.     

Two natural indole glucosinolates from Brassicaceae

Four indole glucosinolates were isolated from different cruciferous tissues and tissue cultures. After separation in the form of their desulfo derivatives, they were separated by HPLC and analysed by mass spectrometry. Besides 3-indolylmethylglucosinolate and 1-methoxy-3-indolylmethylglucosinolate, two new compounds were identified as 5-hydroxy-3-indolylmethylglucosinolate and 5-methoxy-3-indolylmethylglucosinolate, respectively. Feeding experiments revealed that [ring-2-¹⁴C]tryptophan was a precursor of all of these glucosinolates and that [Me-¹⁴C]methionine serves as the methyl donor of the methoxylated derivatives.     

Advanced nanoscale separations and mass spectrometry for sensitive high-throughput proteomics

Recent developments in combined separations with mass spectrometry for sensitive and high-throughput proteomic analyses are reviewed herein. These developments primarily involve high-efficiency (separation peak capacities of ~10³) nanoscale liquid chromatography (flow rates extending down to approximately 20 nl/min at optimal liquid mobile-phase separation linear velocities through narrow packed capillaries) in combination with advanced mass spectrometry and in particular, high-sensitivity and high-resolution Fourier transform ion cyclotron resonance mass spectrometry. Such approaches enable analysis of low nanogram level proteomic samples (i.e., nanoscale proteomics) with individual protein identification sensitivity at the low zeptomole level. The resultant protein measurement dynamic range can approach 10⁶ for nanogram-sized proteomic samples, while more abundant proteins can be detected from subpicogram-sized (total) proteome samples. These qualities provide the foundation for proteomics studies of single or small populations of cells. The instrumental robustness required for automation and providing high-quality routine performance nanoscale proteomic analyses is also discussed.     

Simultaneous quantification of free nucleotides in complex biological samples using ion pair reversed phase liquid chromatography isotope dilution tandem mass spectrometry

A new sensitive and accurate analytical method has been developed for quantification of intracellular nucleotides in complex biological samples from cultured cells of different microorganisms such as Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. This method is based on ion pair reversed phase liquid chromatography electrospray ionization isotope dilution tandem mass spectrometry (IP-LC-ESI-ID-MS/MS. A good separation and low detection limits were observed for these compounds using dibutylamine as volatile ion pair reagent in the mobile phase of the LC. Uniformly ¹³C-labeled isotopes of nucleotides were used as internal standards for both extraction and quantification of intracellular nucleotides. The method was validated by determining the linearity, sensitivity, and repeatability.     

Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels

Ceramide-1-phosphate (C1P) is a bioactive sphingolipid with roles in several biological processes. Currently, high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC ESI-MS/MS) offers the most efficient method of quantifying C1P. However, the published protocols have several drawbacks causing overestimations and carryovers. Here, the reported overestimation of C1P was shown to be due to incomplete neutralization of base hydrolyzed lipid extracts leading to the hydrolysis of SM to C1P. Actual quantity of C1P in cells (6 pmols/10⁶ cells) was much lower than previously reported. Also, the major species of C1P produced by ceramide kinase (CERK) was found to be d_18:1/16:0 with a minority of d_18:1/24:1 and d_18:1/24:0. The artifactual production of C1P from SM was used for generating C1Ps as retention time markers. Elimination of carryovers between samples and a 2-fold enhancement in the signal strength was achieved by heating the chromatographic column to 60^°C. The role of ceramide transport protein (CERT) in supplying substrate to CERK was also revalidated using this new assay. Finally, our results demonstrate the presence of additional pathway(s) for generation of the C1P subspecies, d_18:1/18:0 C1P, as well as a significant portion of d_18:1/16:0, d_18:1/24:1, and d_18:1/24:0. In conclusion, this study introduces a much improved and validated method for detection of C1P by mass spectrometry and demonstrates specific changes in the C1P subspecies profiles upon downregulation of CERK and CERT.     

The twin ion technique for detection of metabolites by gas chromatography-mass spectrometry: intermediates in estrogen biosynthesis

[4-¹⁴C + 7-D_0.44]Androstenedione and [4-¹⁴C + 7β-D_0.42]testosterone were prepared. When they were examined by mass spectrometry, the above proportion of deuterium and protium forms resulted in mass spectra in which the molecular ion (M⁺) and (M⁺ + 1) were of equal intensity. Fragment ions that contained deuterium were also twins. When doubly-labeled androstenedione and testosterone were used as substrates for the aromatizing enzymes of human placenta, the mass spectra of metabolites were characteristically labeled and thus readily distinguished from unlabeled material. Metabolites were quantitated by counting ¹⁴C. 17β,19-Dihydroxyandrost-4-en-3-one, 19-hydroxyandrost-4-ene-3,17-dione, 17β-hydroxy-3-oxoandrost-4-en-19-al, 3,17-dioxoandrost-4-en-19-al, estradiol-17β, and estrone were isolated, identified by their mass spectra, and quantitated following incubation of doubly-labeled androstenedione and testosterone with human placental microsomes.     

Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry

The production of biosurfactant by Bacillus subtilis LSFM-05 was carried out using raw glycerol, obtained from a vegetable oil biodiesel plant in Brazil, as the sole carbon source. Production of the biosurfactant was carried out in a 15-L bench-top fermentor and the surfactant was obtained from the foam produced. The crude surfactant was purified by silica gel column chromatography with a yield of 230 mg of the purified biosurfactant per liter of foam. TLC, IR spectroscopy, ¹H and ¹³C NMR and Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization (ESI-FTMS) were used to characterize the purified surfactant. The isolated surfactant was identified as a surfactin lipopeptide. MS/MS data identified the amino acid sequence as GluOMe-Leu-Leu-Asp-Val-Leu-Leu and showed that the fatty acid moiety contained 14 carbons in iso, anteiso or normal configurations. The critical micelle concentration of the C₁₄/Leu₇ surfactin was 70 μM, with emulsification efficiency after 24 h (E₂₄) of 67.6% against crude oil. Raw glycerol represents an abundant and renewable carbon source and provides an opportunity for reducing the cost of biosurfactant production and may add value to biodiesel production by creating new commercial applications for this by-product.     

Application of high-precision isotope ratio monitoring mass spectrometry to identify the biosynthetic origins of proteins

Isotope ratio monitoring (IRM) mass spectrometry was used to measure the relative abundance of stable isotopes in several samples of adult human hemoglobin expressed in E. coli, yeast, and human blood. The results showed significant differences in the distribution of (15)N and (13)C isotopes among hemoglobin samples produced in these organisms. This indicates that IRM mass spectrometry can be used in forensic protein chemistry to identify the origin of protein expression.     

Applying the tools of chemistry (mass spectrometry and covalent modification by small molecule reagents) to the detection of prions and the study of their structure

Prions are molecular pathogens, able to convert a normal cellular prion protein (PrP^C) into a prion (PrP^Sc). The information necessary for this conversion is contained in the conformation of PrP^Sc. Mass spectrometry (MS) and small-molecule covalent reactions have been used to study prions. Mass spectrometry has been used to detect and quantitate prions in the attomole range (10^?18 mole). MS-based analysis showed that both possess identical amino acid sequences, one disulfide bond, a GPI anchor, asparagine-linked sugar antennae, and unoxidized methionines. Mass spectrometry has been used to define elements of the secondary and tertiary structure of wild-type PrP^Sc and GPI-anchorless PrP^Sc. It has also been used to study the quaternary structure of the PrP^Sc multimer. Small molecule reagents react differently with the same lysine in the PrP^C conformation than in the PrP^Sc conformation. Such differences can be detected by Western blot using mAbs with lysine-containing epitopes, such as 3F4 and 6D11. This permits the detection of PrP^Sc without the need for proteinase K pretreatment and can be used to distinguish among prion strains. These results illustrate how two important chemical tools, mass spectrometry and covalent modification by small molecules, are being applied to the detection and structural study of prions. Furthermore these tools are or can be applied to the study of the other protein misfolding diseases such as Alzheimer Disease, Parkinson Disease, or ALS.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Positive and negative tandem mass spectrometric fingerprints of lipids from the halophilic Archaea Haloarcula marismortui

Lipids from the extremely halophilic Archaea, Haloarcula marismortui, contain abundant phytanyl diether phospholipids, namely archaetidic acid (AA), archaetidylglycerol (AG), archaetidylglycerosulfate (AGS), with mainly archaetidylglycerophosphate methyl ester (AGP-Me). These were accompanied by a triglycosyl archaeol (TGA), lacking characteristic sulfate groups. Tandem-mass spectrometry was employed to provide fingerprints for identifying these known lipids, as well as small amounts of unsaturated phospholipids. These contained 3 and 6 double bonds in their archaeol moiety, suggested by negative tandem-MS of intact phospholipids, as indicated by differences between their pseudo-molecular ion and specific fragment ions designated as π₂. The core ether lipids were confirmed by electrospray ionization mass spectrometry (ESI-MS) as 2,3-di-O-phytanyl-sn-glycerol (C20, C20), which gave rise to a precursor-ion at m/z 660 [M+Li]⁺, and its fragment ion at m/z 379 [M+Li]⁺, consistent with mono-O-phytanyl-glycerol. Furthermore, lithiated ions at m/z 654 (MS¹), 379 (MS²) and m/z 648 (MS¹), 373 (MS²), combined with ¹H/¹³C NMR chemical shifts at δ 5.31-121.6 (C2/2′-H2/2′), 5.08-124.9 (C6/6′-H6/6′) and 5.10-126.0 (10/10′-H10/10′) confirmed the presence of unsaturated homologs of archaeol. We carried out a comprehensive study on the lipids present in cells of H. marismortui. We used positive and negative ESI-MS with tandem-MS, which served as a fingerprint analysis for identifying the majority of component lipids.     

An improved scintillation cocktail of high-solubilizing power

A scintillation cocktail containing 25% Triton X-114 in xylene is considered for a broad range of scintillation counting applications. The cocktail gives good counting efficiencies for ³H (47%) and ¹⁴C (93%). It will accept up to 30% (v/v) aqueous sample. The scintillation fluid is also used effectively with samples which are difficult to solubilize, such as the degradation products from the solubilization of polyacrylamide gels. The cocktail can be formulated for less than $2.00 per gallon.