Purification and mechanistic characterisation of two polygalacturonases from Sclerotium rolfsii

Sclerotium rolfsii (strain CBS 350.80) was found to produce extraordinary high amounts of polygalacturonases (PGs). Two of these extracellular enzymes were purified by a recently introduced preparative electrophoretic device (isoelectric focusing mode of free flow electrophoresis). PG 1 (39.5 kDa, pI 6.5) and PG 2 (38 kDa, pI 5.4) exhibited quite similar properties, they were found to be both endo-acting enzymes. Both PGs cleaved penta- and trigalacturonic acid while tetragalacturonic acid was only cleaved when trigalacturonic acid was present. The latter substrate was hydrolysed much faster by PG 2. Both enzymes were active on pectins with different degrees of esterification, they were sensitive towards Ca-cations and not glycosylated. The kinetic properties were measured by viscosimetry with polygalacturonic acid as a substrate. NMR experiments on a model substrate revealed an inverting mechanism of carbohydrate hydrolysis for both enzymes.     

Balance—FFE序列在胆管磁共振检查中的应用

目的：探讨磁共振平衡式稳态自由进动梯度回波序列（Balance—FFE）在胆管疾病中的应用价值。方法：92例胆管病变患者均进行了冠状面的Balance—FFE序列扫描和磁共振胰胆管造影（MRCP）。将Balance-FFE的图像和3DMRCP像及MRCP原始图像对病变的显示率进行X。检验。结果：胆管系统在Balance—FFE序列中呈明显高信号,并能直接显示胆管结石和胆管狭窄,还能显示胆管外病变和胰腺病变,对周围淋巴结的显示也比较清楚。经x^2检验,Balance—FFE序列和MRCP原始像对胆管系统病变的显示没有统计学意义（P〉0．05）,而3DMRCP像对病变的显示能力不如Balance．FFE序列和MRCP原始像,对胆道病变的显示能力具有统计学意义（P〈0．05）。结论：Balance-FFE序列对胆道病变能清晰显示,成像速度快,图像信噪比高,伪影较少,与MRCP结合能提高对胆管系统病变的诊断率,因此Balance-FFE序列应作为胆管疾病磁共振扫描的常规序列,可作为MRCP序列的有效补充。     

Analysis of human plasma proteins: a focus on sample collection and separation using free-flow electrophoresis

Due to ease of accessibility, plasma has become the sample of choice for proteomics studies directed towards biomarker discovery intended for use in diagnostics, prognostics and even in theranostics. The result of these extensive efforts is a long list of potential biomarkers, very few of which have led to clinical utility. Why have so many potential biomarkers failed validation? Herein, we address certain issues encountered, which complicate biomarker discovery efforts originating from plasma. The advantages of stabilizing the sample at collection by the addition of protease inhibitors are discussed. The principles of free-flow electrophoresis (FFE) separation are provided together with examples applying to various studies. Finally, particular attention is given to plasma or serum analysis using multidimensional separation strategies into which the FFE is incorporated. The advantages of using FFE separation in these workflows are discussed.     

Free-flow electrophoresis for top-down proteomics by Fourier transform ion cyclotron resonance mass spectrometry

High-efficiency prefractionation of complex protein mixtures is critical for top-down proteomics, i.e., the analysis of intact proteins by MS. Free-flow electrophoresis (FFE) can be used for IEF to separate proteins within a pH gradient according to their pIs. In an FFE system, this separation is performed entirely in the liquid phase, without the need for particulate chromatographic media, gels, or membranes. Herein, we demonstrated the compatibility of IEF-FFE with ESI-Fourier transform ICR MS (ESI-FTICR-MS) for top-down experiments. We demonstrated that IEF-FFE of intact proteins were highly reproducible between FFE instruments, between laboratories, and between analyses. Applying native (0.2% hydroxypropylmethyl cellulose) IEF-FFE to an enzyme resulted in no decrease in enzyme activity; applying either native or denaturing (8 M urea) IEF-FFE to a four-protein mixture with different pIs resulted in isolation of each protein into separate fractions in a 96-well plate. After desalting, each protein was sequenced by top-down MS/MS. As an application of this technique, chicken erythrocyte histone H2A-IV and its major modified forms were enriched by IEF-FFE. Top-down analysis revealed Lys-5 to be a major acetylation site, in addition to N-terminal acetylation.     

Membrane separations using molecularly imprinted polymers

This review presents an overview on the promising field of molecularly imprinted membranes (MIM). The focus is onto the separation of molecules in liquid mixtures via membrane transport selectivity. First, the status of synthetic membranes and membrane separation technology is briefly summarized, emphasizing the need for novel membranes with higher selectivities. Innovative principles for the preparation of membranes with improved or novel functionality include self-assembly or supramolecular aggregation as well as the use of templates. Based on a detailed analysis of the literature, the main established preparation methods for MIM are outlined: simultaneous membrane formation and imprinting, or preparation of imprinted composite membranes. Then, the separation capability of MIM is discussed for two different types, as a function of their barrier structure. Microporous MIM can continuously separate mixtures based on facilitated diffusion of the template, or they can change their permeability in the presence of the template ("gate effect"). Macroporous MIM can be developed towards molecule-specific membrane adsorbers. Emerging further combinations of molecularly imprinted polymers (MIPs), especially MIP nanoparticles or microgels, with membranes and membrane processes are briefly outlined as well. Finally, the application potential for advanced MIM separation technologies is summarized.     

Three decades of nucleic acid aptamer technologies: Lessons learned,progress and opportunities on aptamer development

Aptamers are short single-stranded nucleic acid sequences capable of binding to target molecules in a way similar to antibodies. Due to various advantages such as prolonged shelf life, low batch to batch variation, low/no immunogenicity, freedom to incorporate chemical modification for enhanced stability and targeting capacity, aptamers quickly found their potential in diverse applications ranging from therapy, drug delivery, diagnosis, and functional genomics to bio-sensing. Aptamers are generated by a process called SELEX. However, the current overall success rate of SELEX is far from being satisfactory, and still presents a major obstacle for aptamer-based research and application. The need for an efficient selection strategy consisting of defined procedures to deal with a wide variety of targets is significantly important. In this work, by analyzing key aspects of SELEX including initial library design, target preparation, PCR optimization, and single strand DNA separation, we provide a comprehensive analysis of individual steps to facilitate researchers intending to develop personalized protocols to address many of the obstacles in SELEX. In addition, this review provides suggestions and opinions for future aptamer development procedures to address the concerns on key SELEX steps, and post-SELEX modifications.     

Two-dimensional separation of human plasma proteins using iterative free-flow electrophoresis

Blood plasma is the most complex human-derived proteome, containing other tissue proteomes as subsets. This proteome has only been partially characterized due to the extremely wide dynamic range of the plasma proteins of more than ten orders of magnitude. Thus, the reduction in sample complexity prior to mass spectrometric analysis is particularly important and alternative separation methodologies are required to more effectively mine the lower abundant plasma proteins. Here, we demonstrated a novel separation approach using 2-D free-flow electrophoresis (FFE) separating proteins and peptides in solution according to their pI prior to LC-MS/MS. We used the combination of sequential protein and peptide separation by first separating the plasma proteins into specific FFE fractions. Tryptic digests of the separated proteins were generated and subsequently separated using FFE. The protein separation medium was optimized to segregate albumin into specific fractions containing only few other proteins. An optimization of throughput for the protein separation reduced the separation time of 1 mL of plasma to approximately 3 h providing sufficient material for digestion and the subsequent peptide separation. Our approach revealed low-abundant proteins (e.g., L-selectin at 17 ng/mL and vascular endothelial-cadherin precursor at 30 ng/mL) and several tissue leakage products, thus providing a powerful orthogonal separation step in the proteomics workflow.