A MS data search method for improved 15N‐labeled protein identification

Quantitative proteomics using stable isotope labeling strategies combined with MS is an important tool for biomarker discovery. Methods involving stable isotope metabolic labeling result in optimal quantitative accuracy, since they allow the immediate combination of two or more samples. Unfortunately, stable isotope incorporation rates in metabolic labeling experiments using mammalian organisms usually do not reach 100%. As a consequence, protein identifications in ¹⁵N database searches have poor success rates. We report on a strategy that significantly improves the number of ¹⁵N‐labeled protein identifications and results in a more comprehensive and accurate relative peptide quantification workflow.     

Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA)

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. ¹⁵N metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of ¹⁵N or ¹⁴N, the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the ¹⁵N supply, which led to a uniform incorporation of ¹⁵N into the whole plant protein complement. The incorporation rate (97.43 ± 0.11%) of ¹⁵N into ¹⁵N-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.44%) and natural abundance of ¹⁵N/¹⁴N-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified ¹⁵N/¹⁴N stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed ¹⁵N-coded and ¹⁴N-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated.     

Ratio‐dependent significance thresholds in reciprocal 15N‐labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment

Metabolic labeling of plant tissues with ¹⁵N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on ¹⁵N/¹⁴N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio‐dependent SD is used to define p‐values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.     

The bactericidal effect of ultraviolet and visible light on Escherichia coli

The bactericidal radiation dosages at specific wavelengths in the ultraviolet (UV)-visible spectrum are not well documented. Such information is important for the development of new monochromatic bactericidal devices to be operated at different wavelengths. In this study, radiation dosages required to cause mortality of an Escherichia coli strain, ATCC 25922, at various wavelengths between 250 and 532 nm in the UV and visible spectrum were determined. Radiation at 265 nm in the UV region was most efficient in killing the E. coli cells and 100% mortality was achieved at a dose of 1.17 log mJ/cm(2). In the visible spectrum, the radiation dosages required for a one-log reduction of the E. coli cell density at 458 and 488 nm were 5.5 and 6.9 log mJ/cm(2), respectively. However, at 515 and 532 nm, significant killing was not observed at radiation dosage up to 7 log mJ/cm(2). Based on the cell survival data at various radiation dosages between 250 and 488 nm, a predictive equation for the survival of E. coli cells is derived, namely log(S/S(0)) = -(1.089 x 10(7) e(-0.0633lambda))D. The symbols, S(0), S, lambda, and D, represent initial cell density, cell density after irradiation, wavelength of the radiation and radiation dosage, respectively. The proportion of the surviving E. coli cells decreases exponentially with the increase in radiation dosage at a given wavelength. In addition, the radiation dose required for killing a certain fraction of the E. coli cells increases exponentially as the wavelength of radiation increases.     

A theory for 15N/14N fractionation in nitrate-grown vascular plants

We present a theory describing how the δ¹⁵N values of the nitrogen (N) pools in a vascular plant depend on that of its source N (nitrate), on ¹⁵N/¹⁴N fractionations during N assimilation, and on N transport within and N loss from the plant. The theory allows measured δ¹⁵N values to be interpreted in terms of physiological processes. The δ¹⁵N values of various N pools are calculated using three rules: (1) when a pool divides without transformation, there is no change in the δ¹⁵N values of the N entering the resulting pools; (2) when nitrate is assimilated by nitrate reductase, the δ¹⁵N values of the resulting pools (product and residual substrate) are described by a Rayleigh equation; (3) when two N pools mix, the δ¹⁵N value of the mixture is a weighted average of the δ¹⁵N values of the component pools. The theory is written as a spreadsheet and solved numerically. Potentially, it has multiple solutions. Some contravene physiological reality and are rejected. The remainder are distinguished, where possible, using additional physiological information. The theory simulated independent measurements of δ¹⁵N in N pools of Brassica campestris L. var. rapa (komatsuna) and Lycopersicon esculentum Mill. cv. T-5 (tomato). Received: 27 October 1997 / Accepted: 13 January 1998     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

Metabolic engineering of Escherichia coli for the production of cadaverine: A five carbon diamine

A five carbon linear chain diamine, cadaverine (1,5‐diaminopentane), is an important platform chemical having many applications in chemical industry. Bio‐based production of cadaverine from renewable feedstock is a promising and sustainable alternative to the petroleum‐based chemical synthesis. Here, we report development of a metabolically engineered strain of Escherichia coli that overproduces cadaverine in glucose mineral salts medium. First, cadaverine degradation and utilization pathways were inactivated. Next, L ‐lysine decarboxylase, which converts L ‐lysine directly to cadaverine, was amplified by plasmid‐based overexpression of the cadA gene under the strong tac promoter. Furthermore, the L ‐lysine biosynthetic pool was increased by the overexpression of the dapA gene encoding dihydrodipicolinate synthase through the replacement of the native promoter with the strong trc promoter in the genome. The final engineered strain was able to produce 9.61 g L⁻¹ of cadaverine with a productivity of 0.32 g L⁻¹ h⁻¹ by fed‐batch cultivation. The strategy reported here should be useful for the bio‐based production of cadaverine from renewable resources. Biotechnol. Bioeng. 2011; 108:93–103. © 2010 Wiley Periodicals, Inc.     

The regulation of porin expression in Escherichia coli: effect of turgor stress

The OmpF and OmpC porins are major outer membrane proteins of Escherichia coli. Their expression is affected by medium osmolarity such that OmpF is normally produced at low osmolarity and OmpC at high osmolarity. Potassium ion accumulation is a major means by which cells maintain their internal osmolarity in high osmolarity medium in the absence of organic osmolytes such as glycine-betaine. Starvation for potassium causes cells to become turgor stressed. The effect of turgor stress and potassium ion concentration on OmpF and OmpC expression was examined. It was found that ompF gene expression was switched off by turgor stress but there was no concomitant increase in OmpC. Instead, ompC expression responded to the accumulation of potassium ions by the cell in high osmolarity medium.     

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: An application to the production of a 15N-enriched fragment of lactoferrin

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21–31 of human bactericidal protein lactoferrin.     

东北森林两种典型阔叶树种子和幼苗对15N同位素的富集响应

以东北森林两种典型的阔叶树种风力传播种子——花曲柳和色木槭种子为研究对象,通过室内¹⁵N尿素浸泡试验和温室盆栽试验,设置4个浓度(0、0.05、0.1和0.2 g·L^-1)、3个浸泡时间(4、8和12 d)与4个叶期(2、4、6和8叶)处理,研究种子浸泡浓度、浸泡时间和幼苗叶期对种子和幼苗¹⁵N富集的影响.结果表明: 浸泡浓度和浸泡时间对两树种种子δ¹⁵N值均有显著的正反馈作用,高浓度和长时间(0.2 g·L^-1+12 d)更有利于种子¹⁵N总量的富集,花曲柳和色木槭种子¹⁵N同位素最大富集倍数的浸泡浓度和浸泡时间组合分别为0.1 g·L^-1+(4、8 d)和0.05 g·L^-1+(4、8 d);δ¹⁵N值稀释率随幼苗株高的增加先急剧减少(2~6叶)后趋于稳定,幼苗从8叶开始叶片¹⁵N总量降低,表明6叶幼苗更适合追踪幼苗的来源;幼苗叶片δ¹⁵N值与种子浸泡浓度、浸泡时间和种子的δ¹⁵N值呈显著正相关.花曲柳和色木槭种子及幼苗均能成功富集到¹⁵N信号,采用0.1 g·L^-1+8 d+6叶组合最适合追踪花曲柳和色木槭种子和幼苗.     

利用15N示踪技术研究木荷与马尾松幼苗叶片对NO2的吸收与分配

大气氮氧化物(NO_x=NO+NO₂)随着干沉降进入森林生态系统时,会首先接触森林冠层。森林乔木能通过叶片吸收多少NO₂以及对吸收的NO₂是如何分配的,目前尚不清楚。该研究利用¹⁵N稳定同位素示踪技术,对中国南方常见乔木树种木荷(Schima superba)和马尾松(Pinus massoniana)幼苗在黑暗和光照两种条件下进行了¹⁵NO₂静态箱熏蒸实验,检测并分析了两种植物的¹⁵N回收率以及吸收的NO₂在植物各组织中的分配结果。结果显示:植物主要通过气孔吸收NO₂,木荷和马尾松在黑暗条件下整体分别能回收10.3%±5.9%和20.4%±7.0%¹⁵NO₂,在光照条件下整体分别能回收35.9%±5.4%和68.2%±7.6%¹⁵NO₂。两种植物各组织中的平均干质量¹⁵     

From coarse to fine: the absolute Escherichia coli proteome under diverse growth conditions

Accurate measurements of cellular protein concentrations are invaluable to quantitative studies of gene expression and physiology in living cells. Here, we developed a versatile mass spectrometric workflow based on data‐independent acquisition proteomics (DIA/SWATH) together with a novel protein inference algorithm (xTop). We used this workflow to accurately quantify absolute protein abundances in Escherichia coli for > 2,000 proteins over > 60 growth conditions, including nutrient limitations, non‐metabolic stresses, and non‐planktonic states. The resulting high‐quality dataset of protein mass fractions allowed us to characterize proteome responses from a coarse (groups of related proteins) to a fine (individual) protein level. Hereby, a plethora of novel biological findings could be elucidated, including the generic upregulation of low‐abundant proteins under various metabolic limitations, the non‐specificity of catabolic enzymes upregulated under carbon limitation, the lack of large‐scale proteome reallocation under stress compared to nutrient limitations, as well as surprising strain‐dependent effects important for biofilm formation. These results present valuable resources for the systems biology community and can be used for future multi‐omics studies of gene regulation and metabolic control in E. coli.     

Extension of cell membrane boosting squalene production in the engineered Escherichia coli

Squalene is a lipophilic and non-volatile triterpene with many industrial applications for food, pharmaceuticals, and cosmetics. Metabolic engineering focused on optimization of the production pathway suffer from little success in improving titers because of a limited space of the cell membrane accommodating the lipophilic product. Extension of cell membrane would be a promising approach to overcome the storage limitation for successful production of squalene. In this study, Escherichia coli was engineered for squalene production by overexpression of some membrane proteins. The highest production of 612 mg/L was observed in the engineered E. coli with overexpression of Tsr, a serine chemoreceptor protein, which induced invagination of inner membrane to form multilayered structure. It was also observed an increase in unsaturated fatty acid in membrane lipids composition, suggesting cellular response to maintain membrane fluidity against squalene accumulation in the engineered strain. This study potentiates the capability of E. coli for squalene production and provides an effective strategy for the enhanced production of such compounds.     

Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli

Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20 g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions.     

异源表达多巴脱羧酶促进大肠杆菌从头合成多巴胺

多巴胺是多种天然抗氧化药物生物合成的前体物质,在人体内作为神经递质调控中枢神经系统的多种生理功能,常用于多种类型休克的临床治疗.目前,通过微生物合成技术已经实现了多巴胺的从头合成,但是合成效率很低.针对该问题,在左旋多巴(L-DOPA)大肠杆菌工程菌基础上,利用不同拷贝数质粒表达野猪Sus scrofa来源的多巴脱羧酶...