Power and limitations of electrophoretic separations in proteomics strategies

Proteomics can be defined as the large-scale analysis of proteins. Due to the complexity of biological systems, it is required to concatenate various separation techniques prior to mass spectrometry. These techniques, dealing with proteins or peptides, can rely on chromatography or electrophoresis. In this review, the electrophoretic techniques are under scrutiny. Their principles are recalled, and their applications for peptide and protein separations are presented and critically discussed. In addition, the features that are specific to gel electrophoresis and that interplay with mass spectrometry (i.e., protein detection after electrophoresis, and the process leading from a gel piece to a solution of peptides) are also discussed

Capillary and microchip gel electrophoresis using multiplexed fluorescence detection with both time-resolved and spectral-discrimination capabilities: applications in DNA sequencing using near-infrared fluorescence

Increasing the information content obtainable from a single assay and system miniaturization has continued to be important research areas in analytical chemistry. The research presented in this dissertation involves the development of a two-color, time-resolved fluorescence microscope for the acquisition of both steady-state and time-resolved data during capillary and microchip electrophoresis. The utility of this hybrid fluorescence detector has been demonstrated by applying it to DNA sequencing applications. Coupling color discrimination with time-resolved fluorescence offers increased multiplexing capabilities because the lifetime data adds another layer of information. An optical fiber-based fluorescence microscope was constructed, which utilized fluorescence in near-IR region, greatly simplifying the hardware and allowing superior system sensitivity. Time-resolved data was processed using electronics configured in a time-correlated single photon counting format. Cross-talk between color channels was successfully eliminated by utilizing the intrinsic time-resolved capability associated with the detector. The two-color, time-resolved microscope was first coupled to a single capillary and carried out two-color, two-lifetime sequencing of an M13 template, achieving a read length of 650 bps at a calling accuracy of 95.1%. The feasibility of using this microscope with microchips (glass-based chips) for sequencing was then demonstrated. Results from capillaries and microchips were compared, with the microchips providing faster analysis and adequate electrophoretic performance. Lifetimes of a set of fluorescent dyes were determined with favorable precision, in spite of the low loading levels associated with the microchips. The sequencing products were required to be purified and concentrated prior to electrophoretic sorting to improve data quality. PMMA-based microchips for DNA sequencing application were evaluated. The microchips were produced from thermo plastics, which allowed rapid and inexpensive production of microstructures with high aspect ratios. It was concluded that surface coating was needed on the polymer chips in order to achieve single-base resolution required for DNA sequencing. The capability of the two-color time-resolved microscope operated in a scanning mode was further explored. The successful construction of the scanner allows scanning of multi-channel microchips for high throughput processing

Reversible Phospholipid Nanogels: An Alternative Matrix for Tunable High Resolution DNA Sieving with Enhanced Separation Efficiency and Accurate Extended Range Sizing by Capillary Gel Electrophoresis

In an aqueous solution the phospholipids dimyristoyl-sn-glycero-3- phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) self-assemble to form thermo-responsive non-Newtonian fluids (i.e., pseudo-gels) in which small temperature changes of 5-6 °C decrease viscosity dramatically. This characteristic is useful for sieving-based electrophoretic separations (e.g., of DNA), as the high viscosity of linear sieving additives, such as linear polyacrylamide or polyethylene oxide, hinders the introduction and replacement of the sieving agent in microscale channels. While a practical advantage of utilizing phospholipid pseudo-gels for sieving is the ease with which they are introduced into the separation capillary, the separation performance of the material for DNA analyses is exceptional. Capillary electrophoresis separations of DNA are achieved with separation efficiencies ranging from 400,000 to 7,000,000 theoretical plates in a 25 micrometer inner diameter fused silica capillary. Assessment of the phospholipid nanogel with a Ferguson plot yields an apparent pore size of ~31 nm. Under isothermal conditions, Ogston sieving is achieved for DNA fragments smaller than 500 base pairs, whereas reptation-based transport occurs for DNA fragments larger than 500 base pairs. Single base resolution of short tandem repeats relevant to human identification is accomplished with 30 minute separations using traditional capillary electrophoresis instrumentation. Applications that do not require single base resolution are completed with faster separation times. This is demonstrated for a multiplex assay of biallelic single nucleotide polymorphisms relevant to warfarin sensitivity. The thermo-responsive pseudo-gel preparation described here provides a new innovation to sieving based capillary separations. Specific DMPC-DHPC medium is developed to effectively separate and size DNA fragments up to 1,500 base pairs by decreasing the total lipid concentration to 2.5%. A 2.5% phospholipid nanogel generates a resolution of 1% of the DNA fragment size up to 1,500 base pairs. This separation additive is used to evaluate size markers ranging between 200 and 1,500 base pairs in order to distinguish invasive strains of Streptococcus pyogenes and Aspergillus species by harnessing differences in gene sequences of collagen-like proteins in these organisms. For the first time, a reversible stacking gel is integrated in a capillary sieving separation by utilizing the thermally-responsive viscosity of these selfassembled phospholipid preparations. A discontinuous matrix is created that is composed of a cartridge of highly viscous phospholipid assimilated into a separation matrix of low viscosity. DNA sample stacking is facilitated with longer injection times without sacrificing separation efficiency

Manual on application of molecular tools in aquaculture and inland fisheries management. Part 2. Laboratory protocols and data analysis

The aim of this manual is to provide a comprehensive practical tool for the generation and analysis of genetic data for subsequent application in aquatic resources management in relation to genetic stock identification in inland fisheries and aquaculture. The material only covers general background on genetics in relation to aquaculture and fisheries resource management, the techniques and relevant methods of data analysis that are commonly used to address questions relating to genetic resource characterisation and population genetic analyses. No attempt is made to include applications of genetic improvement techniques e.g. selective breeding or producing genetically modified organisms (GMOs). The manual includes two ‘stand-alone’ parts, of which this is the second volume: Part 1 – Conceptual basis of population genetic approaches: will provide a basic foundation on genetics in general, and concepts of population genetics. Issues on the choices of molecular markers and project design are also discussed. Part 2 – Laboratory protocols, data management and analysis: will provide step-by-step protocols of the most commonly used molecular genetic techniques utilised in population genetics and systematic studies. In addition, a brief discussion and explanation of how these data are managed and analysed is also included. This manual is expected to enable NACA member country personnel to be trained to undertake molecular genetic studies in their own institutions, and as such is aimed at middle and higher level technical grades. The manual can also provide useful teaching material for specialised advanced level university courses in the region and postgraduate students. The manual has gone through two development/improvement stages. The initial material was tested at a regional workshop and at the second stage feedback from participants was used to improve the contents

Capillary and microdevice electrophoretic tools for genetic analyses: heteroduplex analysis for tuberculosis drug susceptibility and ligase detection reaction for colorectal cancer detection

The traditional format of electrophoresis, the slab gel, is quickly being replaced by capillary and microdevice platforms, which offer improvements in cost, resolution, speed, quantitation and automation in genetic analyses. These techniques also employ a variety of separation matrices while the slab gel is limited to agarose and polyacrylamide. The research presented here explores the use of these electrophoretic formats for the detection of single nucleotide mutations using two genetic models associated with human disease. Slab gel based heteroduplex analysis (HDA), a popular mutation scanning method, uses a specially designed universal heteroduplex generator (UHG) containing controlled variation to enhance the subtle conformational differences caused by single-base substitutions that are difficult to discriminate. Here, the slab gel based HDA-UHG method has been modified to capillary and microdevice formats for the quantitative and reproducible analysis of single-base substitutions in rpoB that give rise to the rifampin-resistant phenotype of Mycobacterium tuberculosis. The capillary method reduced analysis time from 2.5 hours to approximately 30 minutes. The microdevice further reduced analysis time to 6 minutes while maintaining efficiency and resolution. Both capillary and microdevice methods employed methyl cellulose as the sieving matrix at 0.3% and 0.75% (w/v) concentrations, respectively. In colorectal cancer, base substitutions in the K-ras gene occur early in development, are preserved throughout the course of tumor progression and thus, can be used as biomarkers for the diagnosis of early, curable tumors. The current detection scheme uses slab gel electrophoresis and a mutation specific method, multiplexed polymerase chain reaction/ligase detection reaction, to identify all 19 possible single-base substitution mutations at codons 12, 13 and 61. This technique was also adapted and optimized to the capillary and microdevice formats. This study evaluated capillary methods employing both cross-linked as well as entangled polymer matrices. The capillary methods ranged from 30 to 45 minutes in analysis time. The cross-linked capillary exhibited increased deterioration at longer electrokinetic injection times, while severe injection biases were observed in the entangled polymers evaluated. Initial microdevice experiments were possible in approximately 5 minutes using the entangled polymer matrices and have great potential in microdevice analysis for such mutations

Characterization and mutations in the human sex determining factor SRY

Available from British Library Document Supply Centre-DSC:DXN021834 / BLDSC - British Library Document Supply CentreSIGLEGBUnited Kingdo

Methods of optimization for KCE-based aptamer selection

The success of KCE-based aptamer selection relies on three distinct steps of pre-selection optimization: (i) preventing protein adsorption to the inner walls of the capillary, (ii) maximizing the protein-DNA separation, and (iii) determining an appropriate aptamer-collection window. To perform the first step of optimization, we have developed a simple pressure-based approach which can qualitatively characterize protein adsorption on capillary walls in CE1 o Conceptually, a short plug containing the protein solution is injected into the capillary and carried towards the detection point by applying a low pressure. A dual on-column detector is mimicked by repeating the experiment using the same capillary but a shortened distance to the detection window. The temporal propagation pattern of the protein, at each detection distance, is recorded and the degree of adsorption then analyzed by comparing peak areas and symmetry. This process can be repeated using different buffer additives2, dynamic or permanent coatings until optimal conditions are established. The remaining two steps of pre-selection optimization can be solved by fluorescently labelling the target protein so that its compatibility with KCE separation is maintained3 o By labeling the protein with Chromeo P503, we demonstrate that target detection in CE is markedly improved without significantly affecting the proteins electrophoretic mobility or ability to bind DNA. Thus, Chromeo-labelling can facilitate the accurate identification of both the protein and protein-aptamer complex, which is necessary for maximizing protein-DNA separation and selecting the aptamer collection window. Target-specific considerations must also be optimized prior to commencement of KCE-SELEX. Exonuclease targets, such as Exonuclease I (E.coli), recognize the DNA library as a substrate to be degraded. As a result, the enzyme must be inactivated while still maintaining its native three-dimensional structure during aptamer collection. In addition, non-specific, or unwanted binding at the active site must be avoided, as collection of non-aptamers would limit the progress of KCE-SELEX. For Exol, a divalent metal chelating agent was found to effectively suppress library degradation through experimental optimization. In addition, a small oligonucleotide-based competitive inhibitor was found to bind to the active site with high affinity. This effectively eliminates any unwanted binding of the DNA library at the substrate binding site. The Exol-inhibitor complex can then serve as a target for aptamer development towards an allosteric site of the protein

Cruciform Extrusion in Methylation-State Variants of pBR322 Plasmid

The presence of DNA secondary structures in genomic DNA has been observed in both prokaryotes and eukaryotes. Specifically, sections of ssDNA may melt out and form cruciform/hairpin structures within palindromic sequences or areas of high purine content. Negative supercoiling of the DNA is necessary for energetic favorability of such an event. These noncanonical structures may serve as molecular switches, providing additional regulation of DNA transactions such as transcription and perhaps replication and recombination. Secondary structures associated with regulatory regions are of particular interest as potential sites for the binding of small molecules, which could alter the expression of genes. The effect of system-wide DNA base methylation on extrusion of secondary structures is one aspect of primary-secondary-tertiary structure interplay that has not been examined. Our study first characterized the gross qualitative effect of methylation of plasmid DNA on the formation of cruciform structures. The plasmid pBR322 was transformed into three different strains of E. coli: K12 with functional deoxycytosine and deoxyadenine methyltransferases, BL21 strain (dcm ), and K12 ER2925 with no methyltransferase activity (dam Idem). The resulting purified plasmids were then digested with mung bean nuclease (MBN); a ssDNA-specific nuclease; and then by a site-specific nuclease. The digest reaction mixtures were analyzed by gel electrophoresis for fragments characteristic of cleavage by MBN. The three strains seemed to show no difference in the presence of at least one prominent ssDNA site. Topoisomers of each plasmid were then created by the relaxation of negative supercoils in the presence of DNA intercalator and separated through gel electrophoresis. The three methylation-state variants seemed to exhibit similar structural responses. These data indicate that methylation of respective nucleotide bases does not seem to influence cruciform extrusion, through either changes in local or global conformations

Investigation of electroosmotic flow dynamics and reproducibility in capillary electrophoresis

The goal of this dissertation is to investigate electroosmotic flow (EOF) and electric field dynamics during capillary electrophoresis (CE) experiments using methods based on periodic photobleaching of fluorophores added to the separation buffer at nanomolar concentrations. The methods provide time resolved EOF and local electric field information during an experiment, which can be applied to fundamental studies to provide better understanding of CE techniques. The potential of the EOF monitoring method to improve CE migration reproducibility was investigated in Chapter 2. The EOF monitoring method and four other methods from the literature were applied to the same electrophoretic separations, and their performance for improving reproducibility was compared. The EOF monitoring method significantly improved migration reproducibility, in general; however, much simpler neutral marker method performed nearly well. Biological sample adsorption is a common cause of EOF variability and poor reproducibility for CE. In Chapter 3, the effects of biological samples on EOF dynamics were investigated. Model compounds representing major components of a biological cell and complex biological samples were introduced into the CE system while EOF was monitored continuously. Due to sample adsorption, EOF rates decreased and vacancy peak widths, used for EOF monitoring, increased. It was found that protein molecules had the greatest impact on EOF. Discontinuous solutions in a capillary (zones of different pH, ionic strength or composition) result in generation of different EOF and local electric fields down the length of the capillary. The EOF monitoring method was expanded by adding a charged marker (fluorescein), and this improved method was employed to investigate EOF dynamics and local electric field changes during CE with discontinuous solutions, which were generated by introducing a low ionic strength buffer zone into the capillary. Faster EOF rates in the capillary and faster fluorescein electrophoretic velocities within the sample plug were observed due to high local electric field. Unexpected fluorescein concentration changes were observed during the experiments. These observations led to use of computer simulations in an attempt to understand and reproduce the electrophoresis results. The simulation results, which were obtained using Simul 5.0 indicated the experimental results are consistent with the CE theory

Solid-phase DNA sequencing reactions performed in micro-capillary reactors and solid-phase reversible immobilization in microfluidic chips for purification of dye-labeled DNA sequencing fragments

The research presented in this dissertation involves micro-capillary reactors for solid phase DNA sequencing, the identification of dye terminator sequencing fragments with time-resolved methods, and purification of dye-labeled DNA fragments using solid- phase reversible immobilization in microfluidic chips. Solid-phase micro-reactors have been prepared for DNA sequencing applications using slab gel electrophoresis. A PCR product was immobilized to the interior wall of a fused-silica capillary tube via a biotin-streptavidin linkage. Solid-phase sequencing was carried out in micro-capillary reactors using a four-lane, single color dye primer chemistry strategy. The read length was found to be 589 bases. The complementary DNA fragments generated in the small volume (~62 nL) reactor were directly injected into the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. A set of terminators labeled with near-IR heavy-atom modified tricarbocyanine dyes were investigated for a terminator sequencing protocol in conjunction with slab gel electrophoresis. This protocol gave 605 bp read lengths. A one color, two lifetime format of DNA sequencing was implemented. A pixel-by-pixel analysis was employed to identify each of the bases in the run. The resulting read accuracy for the two-dye capillary run was 90.6%. The use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) was investigated. SPRI cleanup of dye-terminator sequencing fragments using a photoactivated PC microchannel and slab gel electrophoresis produce a read length of 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis system. In this case, the immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for pre-concentration