EZYprep LC‐coupled MALDI‐TOF/TOF MS: An improved matrix spray application for phosphopeptide characterisation

The quality of MALDI‐TOF mass spectrometric analysis is highly dependent on the matrix and its deposition strategy. Although different matrix‐deposition methods have specific advantages, one major problem in the field of proteomics, particularly with respect to quantitation, is reproducibility between users or laboratories. Compounding this is the varying crystal homogeneity of matrices depending on the deposition strategy used. Here, we describe a novel optimised matrix‐deposition strategy for LC‐MALDI‐TOF/TOF MS using an automated instrument that produces a nebulised matrix “mist” under controlled atmospheric conditions. Comparisons of this with previously reported strategies showed the method to be advantageous for the atypical matrix, 2,5‐DHB, and improved phosphopeptide ionisation when compared with deposition strategies for CHCA. This optimised DHB matrix‐deposition strategy with LC‐MALDI‐TOF/TOF MS, termed EZYprep LC, was subsequently optimised for phosphoproteome analysis and compared to LC‐ESI‐IT‐MS and a previously reported approach for phosphotyrosine identification and characterisation. These methods were used to map phosphorylation on epidermal growth factor‐stimulated epidermal growth factor receptor to gauge the sensitivity of the proposed method. EZYprep DHB LC‐MALDI‐TOF/TOF MS was able to identify more phosphopeptides and characterise more phosphorylation sites than the other two proteomic strategies, thus proving to be a sensitive approach for phosphoproteome analysis.     

Newly Betwixt and Between: Revising Liminality in the Context of a Teacher Preparation Program

Through an analysis of a contemporary rite of passage-the final stage of teacher preparation-I develop a new theory of liminality that both builds on and extends Victor Turner's enduring insights. The analysis focuses on how preservice teachers in an undergraduate education program engage in a process of identity formation within an asynchronous, nondimensional, liminal space made possible and shaped by e-mail and with the support of experienced mentor teachers.     

Trends in solventless sample preparation techniques for environmental analysis

The paper presents recent trends in solventless sample preparation techniques for environmental analysis. First, a general classification of solventless methods is given. Next, three of them, treated as preferable techniques, i.e. SPME, SDME and HS, are presented in detail, with respect to their usability and effectiveness for environmental samples. Examples of all discussed techniques are given in the tables.     

Cold glycerol-saline: the promising quenching solution for accurate intracellular metabolite analysis of microbial cells

Microbial metabolomics has been seriously limited by our inability to perform a reliable separation of intra- and extracellular metabolites with efficient quenching of cell metabolism. Microbial cells are sensitive to most (if not all) quenching agents developed to date, resulting in leakage of intracellular metabolites to the extracellular medium during quenching. Therefore, as yet we are unable to obtain an accurate concentration of intracellular metabolites from microbial cell cultures. However, knowledge of the in vivo concentrations of intermediary metabolites is of fundamental importance for the characterization of microbial metabolism so as to integrate meaningful metabolomics data with other levels of functional genomics analysis. In this article, we report a novel and robust quenching method for microbial cell cultures based on cold glycerol-saline solution as the quenching agent that prevents significant leakage of intracellular metabolites and, therefore, permits more accurate measurement of intracellular metabolite concentrations in microbial cells.     

Detection of Salmonella sp. in Dermanyssus gallinae using an FTA filter-based polymerase chain reaction

Salmonella spp. bacteria are responsible for some of the most important zoonoses worldwide. Because Dermanyssus gallinae (DeGeer) (Acari: Dermanyssidae) has been recently reported to be an experimental vector of Salmonella Enteritidis, it would be of benefit to evaluate the presence of this bacterium in mites. A molecular detection tool associating a simple filter-based DNA preparation with a specific 16S rDNA Salmonella sp. polymerase chain reaction (PCR) amplification was described. The limit of detection with this method was 2 x 10(4) bacteria per mite. To adapt this technique for large-scale studies, two sizes of mite pools were tested and a preliminary investigation was carried out on mites from 16 currently or previously contaminated farms. Mites sampled from one farm of each type were positive for Salmonella, suggesting that Dermanyssus could act as a reservoir between flocks. In further investigations, it will be necessary to carry out a large-scale study to assess the role of D. gallinae in the epidemiology of avian salmonellosis.     

Chemical rescue of a site-modified ligand to a [4Fe-4S] cluster in PsaC, a bacterial-like dicluster ferredoxin bound to Photosystem I

Chemical rescue of site-modified amino acids using externally supplied organic molecules represents a powerful method to investigate structure-function relationships in proteins. Here we provide definitive evidence that aryl and alkyl thiolates, reagents typically used for in vitro iron-sulfur cluster reconstitutions, serve as rescue ligands to a site-specifically modified [4Fe-4S]^1+,2+ cluster in PsaC, a bacterial dicluster ferredoxin-like subunit of Photosystem I. PsaC binds two low-potential [4Fe-4S]^1+,2+ clusters termed F_A and F_B. In the C13G/C33S variant of PsaC, glycine has replaced cysteine at position 13 creating a protein that is missing one of the ligating amino acids to iron-sulfur cluster F_B. Using a variety of analytical techniques, including non-heme iron and acid-labile sulfur assays, and EPR, resonance Raman, and Mössbauer spectroscopies, we showed that the C13G/C33S variant of PsaC binds two [4Fe-4S]^1+,2+ clusters, despite the absence of one of the biological ligands. ¹⁹F NMR spectroscopy indicated that the external thiolate replaces cysteine 13 as a substitute ligand to the F_B cluster. The finding that site-modified [4Fe-4S]^1+,2+ clusters can be chemically rescued with external thiolates opens new opportunities for modulating their properties in proteins. In particular, it provides a mechanism to attach an additional electron transfer cofactor to the protein via a bound, external ligand.     

Characterization of the conformational and orientational dynamics of ganglioside GM1 in a dipalmitoylphosphatidylcholine bilayer by molecular dynamics simulations

The structure and dynamics of a single GM1 (Gal5-β1,3-GalNAc4-β1,4-(NeuAc3-α2,3)-Gal2-β1,4-Glc1-β1,1-Cer) embedded in a DPPC bilayer have been studied by MD simulations. Eleven simulations, each of 10 ns productive run, were performed with different initial conformations of GM1. Simulations of GM1-Os in water and of a DPPC bilayer were also performed to delineate the effects of the bilayer and GM1 on the conformational and orientational dynamics of each other. The conformation of the GM1 headgroup observed in the simulations is in agreement with those reported in literature; but the headgroup is restricted when embedded in the bilayer. NeuAc3 is the outermost saccharide towards the water phase. Glc1 and Gal2 prefer a parallel, and NeuAc3, GalNac4 and Gal5 prefer a perpendicular, orientation with respect to the bilayer normal. The overall characteristics of the bilayer are not affected by the presence of GM1; however, GM1 does influence the DPPC molecules in its immediate vicinity. The implications of these observations on the specific recognition and binding of GM1 embedded in a lipid bilayer by exogenous proteins as well as proteins embedded in lipids have been discussed.     

毕赤酵母表达的含前S1、前S2和S表位乙肝表面抗原疫苗的制备和免疫原性研究

整合了乙肝表面抗原嵌合基因SS1和SS2的毕赤酵母工程菌株GS115-SS1S2经高密度发酵培养,甲醇诱导,抗原表达量达到300~600mg/L发酵液。SS1S2抗原经细胞破碎、硅胶吸附、疏水层析和凝胶过滤纯化,纯度达99%以上,每升培养物可收获纯化抗原82mg。纯化的SS1S2抗原经Al(OH)3吸附,在NIH小鼠中进行免疫效果评价。三组NIH雌性小鼠,分别腹腔接种2.5μg、0.625μg和0.156μgSS1S2疫苗或商品化的单S疫苗。部分小鼠在30天时采血,测定各疫苗组的ED50值。在SS1S2疫苗组,前S1、前S2和S抗原的ED50值分别为0.46、0.29和0.84μg,而S疫苗组S抗原的ED50为0.99μg。另一部分小鼠分别在7天和14天时采血,考察抗体阳转率与时间的关系。SS1S2疫苗前S1、前S2抗体阳转率在7d和14d时比S抗体的阳转率为高,提示前S抗体出现的时间较早。上述结果显示SS1S2疫苗比单S疫苗具有更好的免疫原性。     

百日草根尖和花药愈伤组织染色体制片技术

以种子萌发根尖和花药愈伤组织为材料,研究了取样时间、预处理方法对百日草染色体制片的影响。结果表明:根尖上午8:00～9:00,花药愈伤继代3～5d上午9:00～10:00为最佳取样预处理时间;采用三种药剂预处理活体根尖,以4℃下饱和对二氯苯溶液或0.002mol/L的8-羟基喹啉液预处理8h效果最佳,花药愈伤则以饱和对二氯苯溶液预处理6h效果最佳。本实验的预处理温度是固定的,可克服预处理随季节和时间温度的变化而带来的不稳定性,且百日草花药愈伤染色体观察为首次报道。     

一种源于蜗牛的壳聚糖水解酶的纯化和定性

目的研究属于蜗牛的壳聚糖水解酶的纯化方法,得到壳聚糖水解酶的纯品,从而为氨基酸序列分析、基因克隆及工业菌制备奠定前期基础。方法建立检测蜗牛壳聚糖水解酶活性的手段并考察影响酶活性的各种因素,比较现有层析方法纯化蜗牛壳聚糖水解酶的实际效果,确定纯化的最佳条件,从而设计出最合理的纯化方案。结果经苯基琼脂糖柱层析,DEAE-Sepharose离子交换层析和Sephacryl S-300凝胶过滤分离,得到高纯高活性蛋白质,在SDS-PAGE上用银染的方法呈单一蛋白质条带,比活性提高33.333倍,纯化倍数为18.272,得率为0.15。结论实验建立了1种从蜗牛中分离高效高纯度壳聚糖水解酶的方法,为壳寡糖的酶解工业生产提供了新思路、新方法。