A four-column parallel chromatography system for isocratic or gradient LC/MS analyses

A novel approach to parallel liquid chromatography/ tandem mass spectrometry (LC/MS/MS) analyses for pharmacokinetic assays and for similar quantitative applications is presented. Modest modifications render a conventional LC/MS system capable of analyzing samples in parallel. These modifications involve the simple incorporation of three valves and four LC columns into a conventional system composed of one binary LC pumping system, one autosampler, and one mass spectrometer. An increase in sample throughput is achieved by staggering injections onto the four columns, allowing the mass spectrometer to continuously analyze the chromatographic window of interest Using this approach, the optimized run time is slightly greater than the sum of the widths of the desired peaks. This parallel chromatography unit can operate under both gradient and isocratic LC conditions. To demonstrate the utility of the system, atorvastatin, five of its metabolites, and their deuterated internal standards (IS) were analyzed using gradient elution chromatography conditions. The results from a prestudy assay evaluation (PSAE) tray of standards and quality control (QC) samples from extracted spiked human plasma are presented. The relative standard deviation and the accuracy of the QC samples did not exceed 8.1% and 9.6%, respectively, which is well within the acceptance criteria of the pharmaceutical industry. For this particular analysis, the parallel chromatography system decreased the overall run time from 4.5 to 1.65 min and, therefore, increased the overall throughput by a factor of 2.7 in comparison to a conventional LC/MS/MS analytical method.     

Fully automated two-dimensional electrophoresis system for high-throughput protein analysis

We developed a fully automated electrophoresis system for rapid and highly reproducible protein analysis. All the two-dimensional (2D) electrophoresis procedures including isoelectric focusing (IEF), on-part protein staining, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and in situ protein detection were automatically completed. The system comprised Peltiert devices, high-voltage generating devices, electrodes, and three disposable polymethylmethacrylate (PMMA) parts for IEF, reaction chambers, and SDS-PAGE. Because of miniaturization of the IEF part, rapid IEF was achieved in 30 min. A gel with a tapered edge gel on the SDS-PAGE part realized a connection between the parts without use of a gluing material. A biaxial conveyer was employed for the part relocation, sample introduction, and washing processes to realize a low-maintenance and cost-effective automation system. Performances of the system and a commercial minigel system were compared in terms of detected number, resolution, and reproducibility of the protein spots. The system achieved high-resolution comparable to the minigel system despite shorter focusing time and smaller part dimensions. The resulting reproducibility was better or comparable to the performance of the minigel system. Complete 2D separation was achieved within 1.5 h. The system is practical, portable, and has automation capabilities.     

Fully automated chip-based mass spectrometry for complex carbohydrate system analysis

Carbohydrates represent a major class of biopolymers, which occur in nature either as oligosaccharides or glycoconjugates, in which the sugar moiety is linked to proteins or lipids. The significance of mass spectrometry for highly sensitive analysis of complex carbohydrates increased after the introduction of the electrospray ionization and matrix assisted laser desorption/ionization methods and the possibility of tandem MS for sequencing of single molecular species in complex mixtures. Rapid and sensitive characterization of carbohydrates in biological systems by automated nanoscale liquid delivery and chip-based electrospray interface techniques have not been developed so far. In this contribution, the implementation and optimization of a fully automated chip-based nanoelectrospray assembly (NanoMate system), operating in the negative ion mode, in combination with QTOF-tandem MS for mapping/sequencing and computer-assisted structure assignment for carbohydrate components in complex mixtures is presented.     

Maximization of the throughput of a computerized automated warehousing- system under system constraints

This paper attempts to determine the maximum number of storage and retrieval requests that can be handled by automated warehousing systems under physical and operational constraints. The system is formulated as a stochastic constrained optimization problem and an algorithm is developed for its solution. Due to the stochastic nature of the operation of the system, a simulation model is constructed to evaluate the system for various values of its parameters. The optimization routine interacts with the simulation model and systematically moves towards the optimum solution. The problem is studied under various levels of constraints. Also several levels of the risk of violation of constraints are examined.     

Fully automated single-molecule force spectroscopy for screening applications

With the introduction of single-molecule force spectroscopy (SMFS) it has become possible to directly access the interactions of various molecular systems. A bottleneck in conventional SMFS is collecting the large amount of data required for statistically meaningful analysis. Currently, atomic force microscopy (AFM)-based SMFS requires the user to tediously 'fish' for single molecules. In addition, most experimental and environmental conditions must be manually adjusted. Here, we developed a fully automated single-molecule force spectroscope. The instrument is able to perform SMFS while monitoring and regulating experimental conditions such as buffer composition and temperature. Cantilever alignment and calibration can also be automatically performed during experiments. This, combined with in-line data analysis, enables the instrument, once set up, to perform complete SMFS experiments autonomously.     

Nanopore-based proteolytic reactor for sensitive and comprehensive proteomic analyses

Various silica-based microreactors have been designed that use enzyme immobilization to address technical concerns in proteolysis including inefficient and incomplete protein digestion. Most of current designs for proteolytic reactors can improve either protease stability or proteolysis efficiency of individual protein(s). However, the desired features such as rapid digestion, larger sequence coverage, and high sensitivity have not been achieved by a single microreactor design for broad range proteins with diverse physical properties. Here, unlike conventional enzyme immobilization strategies, we describe a novel proteolytic nanoreactor based on the unique three-dimensional nanopore structure of our newly synthesized mesoporous silica (MPS), FDU-12, which integrates substrate enrichment, "reagent-free" protein denaturation, and efficient proteolytic digestion. In our design, protein substrates were first captured by MPS nanopore structure and were concentrated from the solution. Following the pH change and applying trypsin, the denaturation and concurrent proteolysis of broad-range proteins were efficiently achieved. In minutes, many more sample peptides from the in-nanopore digestion of protein mixtures were detected by mass spectrometry, resulting in the identifications of a broad range of diverse proteins with high sequence coverage. The unique features of FDU-12 nanostructure that allow rapid, complete proteolysis and resulting enhanced sequence coverage of individual proteins were investigated by using Raman spectroscopy and comparative studies with respect to other MPSs.     

Integration of two-dimensional LC-MS with multivariate statistics for comparative analysis of proteomic samples

LC-MS-based proteomics requires methods with high peak capacity and a high degree of automation, integrated with data-handling tools able to cope with the massive data produced and able to quantitatively compare them. This paper describes an off-line two-dimensional (2D) LC-MS method and its integration with software tools for data preprocessing and multivariate statistical analysis. The 2D LC-MS method was optimized in order to minimize peptide loss prior to sample injection and during the collection step after the first LC dimension, thus minimizing errors from off-column sample handling. The second dimension was run in fully automated mode, injecting onto a nanoscale LC-MS system a series of more than 100 samples, representing fractions collected in the first dimension (8 fractions/sample). As a model study, the method was applied to finding biomarkers for the antiinflammatory properties of zilpaterol, which are coupled to the beta2-adrenergic receptor. Secreted proteomes from U937 macrophages exposed to lipopolysaccharide in the presence or absence of propanolol or zilpaterol were analysed. Multivariate statistical analysis of 2D LC-MS data, based on principal component analysis, and subsequent targeted LC-MS/MS identification of peptides of interest demonstrated the applicability of the approach.     

Fully integrated on-chip electrochemical detection for capillary electrophoresis in a microfabricated device

Microfabricated lab-on-a-chip devices employing a fully integrated electrochemical (EC) detection system have been developed and evaluated. Both capillary electrophoresis (CE) channels and all CE/EC electrodes were incorporated directly onto glass substrates via traditional microfabrication techniques, including photolithographic patterning, wet chemical etching, DC sputtering, and thermal wafer bonding. Unlike analogous CE/EC devices previously reported, no external electrodes were required, and critical electrode characteristics, including size, shape, and placement on the microchip, were established absolutely by the photolithography process. For the model analytes dopamine and catechol, detection limits in the 4-5 microM range (approximately 200 amol injected) were obtained with the Pt EC electrodes employed here, and devices gave stable analytical performance over months of usage.     

Mass defect labeling of cysteine for improving peptide assignment in shotgun proteomic analyses

A method for improving the identification of peptides in a shotgun proteome analysis using accurate mass measurement has been developed. The improvement is based upon the derivatization of cysteine residues with a novel reagent, 2,4-dibromo-(2'-iodo)acetanilide. The derivitization changes the mass defect of cysteine-containing proteolytic peptides in a manner that increases their identification specificity. Peptide masses were measured using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron mass spectrometry. Reactions with protein standards show that the derivatization of cysteine is rapid and quantitative, and the data suggest that the derivatized peptides are more easily ionized or detected than unlabeled cysteine-containing peptides. The reagent was tested on a 15N-metabolically labeled proteome from M. maripaludis. Proteins were identified by their accurate mass values and from their nitrogen stoichiometry. A total of 47% of the labeled peptides are identified versus 27% for the unlabeled peptides. This procedure permits the identification of proteins from the M. maripaludis proteome that are not usually observed by the standard protocol and shows that better protein coverage is obtained with this methodology.     

Fully automated mixed mode crack propagation analyses based on tetrahedral finite element and VCCM (virtual crack closure-integral method)

The authors have been developing a crack propagation analysis system that can deal with arbitrary shaped cracks in three-dimensional solids. The system is consisting of mesh generation software, a large-scale finite element analysis program and a fracture mechanics module. To evaluate the stress intensity factors, virtual crack closure-integral method (VCCM) for the quadratic tetrahedral finite element is adopted and is included in the fracture mechanics module. The rate and direction of crack propagation are predicted by using appropriate formulae based on the stress intensity factors. In this paper, the crack propagation system is briefly described and some numerical results are presented.     

An automated finite element procedure for fatigue crack propagation analyses

This paper presents an innovative approach for mixed-mode two-dimensional fatigue crack propagation analyses of structural components via interactive computer graphics. Linear elastic fracture mechanics and the finite element method are integrated in a comprehensive computer code that determines the crack path and fatigue life for a given problem. In addition, the program has the capability of automatic finite element remeshing at any arbitrary location, angle or direction following crack nucleation or extension in a continuously monitored analysis. To demonstrate the proficiency of the program, a study was performed that investigated the fatigue life of attachment lugs. Correlation with existing experimental data and other numerical procedures verifies the accuracy of the program.     

Plant-level maintenance decision support system for throughput improvement

In the current global scenario of extreme competition, factors such as productivity, availability, quality and cost of operations play a vital role in the success of a company. A critical component relating to all of the above is maintenance. The conventional maintenance decision support systems have primarily focused on maximising the gains of a single machine system. However, a real life application usually consists of multiple machines, and the operational level decisions are more complex. In this paper, an on line plant-level maintenance decision support system (PMDSS) is developed by combining the short term and long term decision making process to improve the overall system performance while continuously attempting to maximise immediate profits in the short term. The PMDSS works towards two basic aims: (1) unplanned downtime reduction by predicting the remaining useful life of the machines, and (2) efficient utilisation of the finite maintenance and production resources through identifying the throughput-critical machines. The benefits of this approach are presented by considering an industrial case study of an automotive assembly line. The results obtained using this PMDSS approach shows a big throughput improvement as compared to the conventional maintenance policies.     

High-performance liquid chromatography-electrospray ionization mass spectrometry using monolithic capillary columns for proteomic studies

The use of tetrahydrofuran/decanol as porogens for the fabrication of micropellicular poly(styrene/divinylbenzene) monoliths enabled the rapid and highly efficient separation of peptides and proteins by reversed-phase high-performance liquid chromatography (RP-HPLC). In contrast to conventional, granular, porous stationary phases, in which the loading capacity is a function of molecular mass, the loadability of the monoliths both for small peptides and large proteins was within the 0.40.9-pmol range for a 60- x 0.2-mm capillary column. Lower limits of detection obtained by measuring UV-absorbance at 214 nm with a 3-nl capillary detection cell were 500 amol for an octapeptide and 200 amol for ribonuclease A. Upon reduction of the concentration of trifluoroacetic acid in the eluent from the commonly used 0.1-0.2 to 0.05%, the separation system was successfully coupled to electrospray ionization mass spectrometry (ESI-MS) at the cost of only a small decrease in separation efficiency. Detection limits for proteins with ESI-MS were in the lower femtomole range. High-quality mass spectra were extracted from the reconstructed ion chromatograms, from which the masses of both peptides and proteins were deduced at a mass accuracy of 50-150 ppm. The applicability of monolithic column technology in proteomics was demonstrated by the mass fingerprinting of tryptic peptides of bovine catalase and human transferrin and by the analysis of membrane proteins related to the photosystem II antenna complex of higher plants.     

Detection of phosphorylated peptides in proteomic analyses using microfluidic compact disk technology

A compact disk (CD)-based microfluidic method for selective detection of phosphopeptides by mass spectrometry is described. It combines immobilized metal affinity chromatography (IMAC) and enzymatic dephosphorylation. Phosphoproteins are digested with trypsin and processed on the CD using nanoliter scale IMAC with and without subsequent in situ alkaline phosphatase treatment. This is followed by on-CD matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Dephosphorylation of the IMAC-enriched peptides allows selective phosphopeptide detection based on the differential mass maps generated (mass shifts of 80 Da or multiples of 80 Da). The CD contains 96 microstructures, each with a 16 nL IMAC microfluidic column. Movement of liquid is controlled by differential spinning of the disk. Up to 48 samples are distributed onto the CD in two equal sets. One set is for phosphopeptide enrichment only, the other for identical phosphopeptide enrichment but combined with in situ dephosphorylation. Peptides are eluted from the columns directly into MALDI target areas, still on the CD, using a solvent containing the MALDI matrix. After crystallization, the CD is inserted into a MALDI mass spectrometer for analysis down to the femtomole level. The average success rate in phosphopeptide detection is over 90%. Applied to noncharacterized samples, the method identified two novel phosphorylation sites, Thr 735 and Ser 737, in the ligand-binding domain of the human mineralocorticoid receptor.     

Techniques for the optimization of proteomic strategies based on head column stacking capillary electrophoresis

Proteomics is the large-scale study of the proteins related to a genome. Presently, proteomic procedures have relied on mass spectrometry as a tool of choice to perform analysis of proteins. Optimization and understanding of the different steps involved in proteomics using mass spectrometry is expensive and time-consuming and, for this reason, have been typically paid insufficient attention. However, optimization becomes a critical issue as we try to analyze ever shrinking amounts of proteins. We present here the development of a technique that allows the rapid, sensitive, semiquantitative, and automated optimization of the processes involved in proteomics. Furthermore, it allows the rapid testing of new methodologies without having to rely on expensive mass spectrometric techniques. The technique, based on head column stacking capillary zone electrophoresis, allows the concentration, separation, and analysis of protein digests at concentrations from high picomoles to subfemtomoles per microliter and sample volumes from a few microliters to a few hundred microliters produced by proteomic processes. Furthermore, the incorporation of UV detection in the system allows the tracking of the relative changes in peptide levels observed during optimization. In addition, all the buffers and solvents used in this technique are compatible with its future coupling to electrospray ionization mass spectrometry. The potential of this technique for the analysis of low-abundance proteins is demonstrated using peptide standards and tryptic digests of standard proteins. Moreover, we exemplify the application of this technique in proteomic prototyping for the rapid and automated study of the procedure of enzymatic digestion of proteins.     

Fully plastic analyses for notched bars and plates using finite element limit analysis

In the present work, fully plastic analyses for notched bars and (plane strain) plates in tension are performed, via finite element (FE) limit analysis based on non-hardening plasticity, from which plastic limit loads and stress fields are determined. Relevant geometric parameters are systematically varied to cover all possible ranges of the notch depth and radius. For the limit loads, it is found that the FE solutions for the notched plate agree well with the existing solution. For the notched bar, however, the FE solutions are found to be significantly different from known solutions, and accordingly the new approximation is given. Regarding fully plastic stress fields, it is found that, for the notched plate, the maximum hydrostatic (mean normal) stress overall occurs in the center of the specimen, which strongly depends on the relative notch depth and the notch radius-to-depth ratio. On the other hand, for the notched bar, the maximum hydrostatic stress can occur in between the center of the specimen and the notch tip. The maximum hydrostatic stress for a given notch depth can occur not for the cracked case, but for the notched case with a certain radius. This is true for both bars and plates. For a given notch radius, on the other hand, the maximum hydrostatic stress increases monotonically with the decreasing notch radius.     

嵌入式系统的U-Boot移植分析

U-Boot软件是一款功能强大的嵌入式系统Bootloader开发工具,Bootloader用来在启动时引导嵌入式系统,是进行嵌入式系统开发的基础.以UP_NETARM3000开发板为基础,结合移植的实践过程,分析了U-Boot移植到三星S3CA480微处理器的详细操作.实验结果表明,移植的U-Boot可以在目标板上成功运行.通过它可以与主机通过串口通信,查看目标板内存,进行操作系统内核及文件系统的烧写等.     

Fully microfabricated and integrated SU-8-based capillary electrophoresis-electrospray ionization microchips for mass spectrometry

We present a fully microfabricated and monolithically integrated capillary electrophoresis (CE)-electrospray ionization (ESI) chip for coupling with high-throughput mass spectrometric (MS) analysis. The chips are fabricated fully of a negative photoresist SU-8 by a standard lithographic process which enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, reproducible analytical performance from chip to chip. As the coaxial sheath flow interface is patterned as an integral part of the SU-8 chip, the fluidic design is dead-volume-free. No significant peak broadening occurs so that very narrow peak widths (down to 2-3 s) are obtained. The sheath flow interface also enables comprehensive optimization of both the CE and the ESI conditions separately so that the same chip design is adaptable to diverse analytical conditions. Plate numbers of the order of 105 m-1 and good resolution are routinely reached for small molecules and peptides within a 2 cm separation length and a typical cycle time of only 30-90 s per sample. In addition, a limit of detection of 100 nM corresponding to a total amount of only 4.5 amol (per injection volume of 45 pL) and excellent quantitative linearity (R2 = 0.9999; 100 nM to 100 microM) were obtained in small-molecule analysis using verapamil as a test compound. The quantitative repeatability was proven good (8.5-21.4% relative standard deviation, peak area) also for the other drug substances and peptides tested.     

Chip-capillary hybrid device for automated transfer of sample preseparated by capillary isoelectric focusing to parallel capillary gel electrophoresis for two-dimensional protein separation

In this article, we introduce a chip-capillary hybrid device to integrate capillary isoelectric focusing (CIEF) with parallel capillary sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) or capillary gel electrophoresis (CGE) toward automating two-dimensional (2D) protein separations. The hybrid device consists of three chips that are butted together. The middle chip can be moved between two positions to reroute the fluidic paths, which enables the performance of CIEF and injection of proteins partially resolved by CIEF to CGE capillaries for parallel CGE separations in a continuous and automated fashion. Capillaries are attached to the other two chips to facilitate CIEF and CGE separations and to extend the effective lengths of CGE columns. Specifically, we illustrate the working principle of the hybrid device, develop protocols for producing and preparing the hybrid device, and demonstrate the feasibility of using this hybrid device for automated injection of CIEF-separated sample to parallel CGE for 2D protein separations. Potentials and problems associated with the hybrid device are also discussed.