PICquant: a quantitative platform to measure differential peptide abundance using dual-isotopic labeling with 12C6- and 13C6-phenyl isocyanate

We have developed a complete system for the isotopic labeling, fractionation, and automated quantification of differentially expressed peptides that significantly facilitates candidate biomarker discovery. We describe a new stable mass tagging reagent pair, (12)C(6)- and (13)C(6)-phenyl isocyanate (PIC), that offers significant advantages over currently available tags. Peptides are labeled predominantly at their amino termini and exhibit elution profiles that are independent of label isotope. Importantly, PIC-labeled peptides have unique neutral-mass losses upon CID fragmentation that enable charge state and label isotope identification and, thereby, decouple the sequence identification from the quantification of candidate biomarkers. To exploit these properties, we have coupled peptide fractionation protocols with a Thermo LTQ-XL LC-MS(2) data acquisition strategy and a suite of automated spectrum analysis software that identifies quantitative differences between labeled samples. This approach, dubbed the PICquant platform, is independent of protein sequence identification and excludes unlabeled peptides that otherwise confound biomarker discovery. Application of the PICquant platform to a set of complex clinical samples showed that the system allows rapid identification of peptides that are differentially expressed between control and patient groups.     

A microfabricated fluidic device for performing two-dimensional liquid-phase separations

A microfabricated fluidic device that combines micellar electrokinetic chromatography and high-speed open-channel electrophoresis on a single structure for the rapid automated two-dimensional analysis of peptides has been devised and demonstrated. The microchip operates by rapidly sampling and analyzing effluent in the second dimension from the first dimension. Second-dimension analyses are performed and completed every few seconds, with total analysis times of less than 10 min for tryptic peptides. The peak capacity of the two-dimensional separations has been estimated to be in the 500-1000 range. The orthogonality of the separation techniques, an important factor for maximizing peak capacity or resolution elements, was verified by examining each technique independently for peptide separations. The two-dimensional separation strategy was found to greatly increase the resolving power over that obtained for either dimension alone.     

On-line immunoaffinity-liquid chromatography-mass spectrometry for identification of amyloid disease markers in biological fluids

Disease-specific alterations in proteins and peptides such as the appearance of new isoforms, changed relative concentrations of known isoforms, or changed catabolism characterize the group of protein precipitation disorders collectively known as amyloidoses. The goal of this study was to develop an approach for isolating and characterizing the pool of isoforms of a polypeptide of interest from biological fluids for use in development of diagnostic markers and elucidation of pathogenesis. For this purpose, we employed an on-line immunoaffinity-liquid chromatography-mass spectrometry (IA-LC-MS) modular approach using antibodies binding populations of protein isoforms. In this system, crude biological samples, e.g., serum, may be injected and subjected to fast hands-off analysis. The setup consists of an optional preclear column for removal of unspecific binding components, an immunoaffinity column, a short cartridgelike reversed-phase column, and an electrospray time-of-flight mass spectrometer. We have tested the system for the automated analysis of three amyloid-related polypeptides, serum amyloid P component, amyloid beta-peptide, and beta2-microglobulin, and we show the feasibility of detection of altered isoforms or determination of relative abundance of isoforms of the proteins from serum or cerebrospinal fluid samples. For each new protein investigated, the only change needed in the system is a new antibody or antibody mixture and the selection of a reversed-phase cartridge of appropriate hydrophobicity.     

Interface for direct and continuous sample-matrix deposition onto a MALDI probe for polymer analysis by thermal field flow fractionation and off-line MALDI-MS

A simple interface based on an oscillating capillary nebulizer (OCN) is described for direct deposition of eluate from a thermal field-flow fractionation (ThFFF) system onto a matrix-assisted laser desorption/ionization (MALDI) probe. In this study, the polymer-containing eluent from the ThFFF system was mixed on-line with MALDI matrix solution and deposited directly onto a moving MALDI probe. The result was a continuous sample track representative of the fractionation process. Subsequent off-line MALDI-mass spectrometry analysis was performed in automated and manual modes. Polystyrene samples of broad polydispersity were used to characterize the overall system performance. The OCN interface is easy to build and operate without the use of heaters or high voltages and is compatible with any MALDI probe format.     

Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion

Changes in protein glycosylation profoundly affect protein function. To understand these effects of altered protein glycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-protein interactions. Methods are not available for amplification of glycans; therefore, highly efficient sample preparation is a major issue. Here we present a novel strategy that allows flexible and sequential incorporation of various functional tags into oligosaccharides derived from biological samples in a practical manner. When combined with a chemoselective glycoblotting platform, our analysis enables us to complete sample preparation (from serum to released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96 samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductive alkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency. Using this technique, we quantitatively detected more than 120 glycans on human carcinoembryonic antigens for the first time. This approach was further developed to include a streamlined method of purification, chromatographic fractionation, and immobilization onto a solid support for interaction analysis. Since our approach enables rapid, flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.     

Microscale isoelectric fractionation using photopolymerized membranes

In this work, we introduce microscale isoelectric fractionation (μIF) for isolation and enrichment of molecular species at any desired location in a microfluidic chip. Narrow pH-specific polyacrylamide membranes are photopatterned in situ for customizable device fabrication; multiple membranes of precise pH are easily incorporated throughout existing channel layouts. Samples are electrophoretically driven across the membranes such that charged species, for example, proteins and peptides, are rapidly discretized into fractions based on their isoelectric points (pI) without the use of carrier ampholytes. This format makes fractions easy to compartmentalize and access for integrated preparative or analytical operations on-chip. We present and discuss the key design considerations and trade-offs associated with proper system operation and optimal run conditions. Efficient and reproducible fractionation of model fluorescent pI markers and proteins is achieved using single membrane fractionators at pH 6.5 and 5.3 from both buffer and Escherichia coli cell lysate sample conditions. Effective fractionation is also shown using a serial 3-membrane fractionator tailored for isolating analytes-of-interest from high abundance components of serum. We further demonstrate that proteins focused in pH specific bins can be rapidly and efficiently transferred to another location in the same chip without unwanted dilution or dispersive effects. μIF provides a rapid and versatile option for integrated sample prep or multidimensional analysis, and addresses the glaring proteomic need to isolate trace analytes from high-abundance species in minute volumes of complex samples.     

Pronase-immobilized enzyme reactor: an approach for automation in glycoprotein analysis by LC/LC-ESI/MSn

An automated analytical approach is proposed for simultaneous characterization of glycan and peptide moieties in pronase-generated glycopeptides. The proposed method is based on the use of a new pronase-immobilized enzyme reactor for the on-line rapid digestion of the target glycoprotein. By coupling the bioreactor to a Hypercarb chromatographic trap column, on-line selective glycopeptide enrichment prior to normal-phase liquid chromatography-mass spectrometry was obtained. A detailed study was carried out for integration and automation of each phase of the proposed analytical procedure. On-line digestion allowed extensive cleavage of the model protein (ribonuclease B), yielding to glycopeptides with peptide moieties up to eight amino acids, carrying the Man5-Man9 N-glycans each, selectively resolved on an Amide-80 column. The use of a linear ion trap instrument resulted in efficient ion capture and led to MS3 acquisition times and spectra quality similar to those for MS2, allowing the unambiguous identification of glycan (MS2) and peptide (MS3) sequences. The proposed procedure reduces the glycoprotein analysis time from approximately 3 days, as in most of the traditional off-line methods, to approximately 1 h.     

Comparison of two-dimensional fractionation techniques for shotgun proteomics

Two-dimensional (2D) fractionation is a commonly used tool to increase dynamic range and proteome coverage for bottom-up, shotgun proteomics. However, there are few reports comparing the relative separation efficiencies of 2D methodologies using low-microgram sample quantities. In order to systematically evaluate 2D separation techniques, we fractionated microgram quantities of E. coli protein extract by seven different methods. The first dimension of separation was performed with either reversed-phase high-pressure liquid chromatography (RP-HPLC), gel electrophoresis (SDS-PAGE), or strong cation exchange (SCX-HPLC). The second dimension consisted of a standard reversed-phase capillary HPLC coupled to an electrospray ionization quadrupole time-of-flight mass spectrometer for tandem mass spectrometric analysis. The overall performance and relative fractionation efficiencies of each technique were assessed by comparing the total number of proteins identified by each method. The protein-level RP-HPLC and the high-pH RP-HPLC peptide-level separations performed the best, identifying 281 and 266 proteins, respectively. The online pH variance SCX and the SDS-PAGE returned modest performances with 178 and 139 proteins identified, respectively. The offline SCX had the worst performance with 81 proteins identified. We also examined various chromatographic factors that contribute to separation efficiency, including resolving power, orthogonality, and sample loss.     

Optimal algorithms for row layout problems in automated manufacturing systems

In many automated manufacturing environments, particularly flowlines and flexible manufacturing systems (FMSs), machines are arranged along a straight material-handling track with a material-handling device moving jobs from one machine to another. These layouts are referred to as row machine layouts. In this paper we study the row layout problem (RLP) under the design objective of minimizing the total backtracking distance of the material-handling device, which is an NP-complete problem. We propose the use of a dynamic programming algorithm for its solution. A special case of the problem, usually encountered in flexible manufacturing cells and which can be solved with a polynomial procedure, is also discussed. For the equidistance case (i.e., successive candidate locations are in equal distances), we formulate the problem as an integer linear program. The use of standard mathematical programming codes can efficiently solve this formulation.     

Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A detailed tryptic map is presented for recombinant human tissue plasminogen activator (rt-PA). Electrospray ionization mass spectrometry is utilized as an on-line HPLC detector for tryptic mapping of this glycoprotein. The additional dimension provided by mass spectrometry gives considerably more detail about the complex tryptic map and significantly enhances the high-resolution chromatographic separation by distinguishing by mass any coeluting components. Through this improvement, the proline isomers of a tryptic peptide were observed eluting over a broad range of retention times. The glycopeptides of rt-PA are observed as well as any corresponding nonglycosylated peptides. In addition, the carbohydrate heterogeneity is readily observed, allowing analysis of the carbohydrate composition. The characteristic diagonal patterns formed by glycopeptides in a contour plot of the data allow rapid recognition of the glycopeptides.     

CAD-based integrated tolerancing system

Assembly tolerance analysis involves the determination of tolerances of critical dimensions. This paper addresses two basic areas in tolerance analysis and allocation. (1) Dimension normalization, which identifies the relevant functional dimensions. A new algorithm called the dimension block diagram (DBD) algorithm for dimension normalization is presented. (2) Dimension representation, which provides a robust framework for representing the tolerance chain. A new dimension representation framework using Assembly Dimensional Tolerance (ADT) tree is presented. The two methodologies are automated and integrated within the framework of a CAD system using an Expert System. A computer test bed software has been developed demonstrating the viability of this approach.     

A systematic approach to determine the optimal maintenance policy for an automated manufacturing system

We propose a systematic approach to determine the optimal maintenance policy for an automated manufacturing system which includes a flexible manufacturing cell (FMC) and several automated machine shops. The systematic approach combines simulation, fractional factorial design, noise or outer array of Taguchi design, regression metamodelling, and classical queueing analysis. A useful expression of the fractional utilization of the manufacturing system is derived and incorporated into formulating and solving the corresponding decision problem. The systematic approach provides an effective implementation procedure to handle practical maintenance problems found in a complex manufacturing environment. © 1997 John Wiley & Sons, Ltd.     

An automated on-line multidimensional HPLC system for protein and peptide mapping with integrated sample preparation

A comprehensive on-line two-dimensional 2D-HPLC system with integrated sample preparation was developed for the analysis of proteins and peptides with a molecular weight below 20 kDa. The system setup provided fast separations and high resolving power and is considered to be a complementary technique to 2D gel electrophoresis in proteomics. The on-line system reproducibly resolved approximately 1000 peaks within the total analysis time of 96 min and avoided sample losses by off-line sample handling. The low-molecular-weight target analytes were separated from the matrix using novel silica-based restricted access materials (RAM) with ion exchange functionalities. The size-selective sample fractionation step was followed by anion or cation exchange chromatography as the first dimension. The separation mechanism in the subsequent second dimension employed hydrophobic interactions using short reversed-phase (RP) columns. A new column-switching technique, including four parallel reversed-phase columns, was employed in the second dimension for on-line fractionation and separation. Gradient elution and UV detection of two columns were performed simultaneously while loading the third and regenerating the fourth column. The total integrated workstation was operated in an unattended mode. Selected peaks were collected and analyzed off-line by MALDI-TOF mass spectrometry. The system was applied to protein mapping of biological samples of human hemofiltrate as well as of cell lysates originating from a human fetal fibroblast cell line, demonstrating it to be a viable alternative to 2D gel electrophoresis for mapping peptides and small proteins.     

CE-microreactor-CE-MS/MS for protein analysis

We present a proof-of-principle for a fully automated bottom-up approach to protein characterization. Proteins are first separated by capillary electrophoresis. A pepsin microreactor is incorporated into the distal end of this capillary. Peptides formed in the reactor are transferred to a second capillary, where they are separated by capillary electrophoresis and characterized by mass spectrometry. While peptides generated from one digestion are being separated in the second capillary, the next protein fraction undergoes digestion in the microreactor. The migration time in the first dimension capillary is characteristic of the protein while migration time in the second dimension is characteristic of the peptide. Spot capacity for the two-dimensional separation is 590. A MS/MS analysis of a mixture of cytochrome c and myoglobin generated Mascot MOWSE scores of 107 for cytochrome c and 58 for myoglobin. The sequence coverages were 48% and 22%, respectively.     

CFRP柔性件保形加工中的变形控制

碳纤维增强树脂基复合材料(CFRP)柔性件的保形加工是航空航天高端装备制造的重要环节,柔性件的可靠装夹是控制加工变形、降低加工尺寸偏差的前提。首先,在理论分析的基础上,明确了柔性件装夹中夹紧及摩擦约束基本条件,提出了基于悬臂梁理论的“随形-就近”吸盘分布原则。进而,使用“ISIGHTABAQUS”联合仿真方法,实现了不同装夹条件及等效切削力作用下CFRP柔性件变形的仿真分析,分析表明：真空吸盘的弹性变形易加大装夹变形,应采用弹性真空吸盘与刚性定位吸盘组合的方式;定位吸盘数量为8、12或16,并“随形-就近”分布时,真空吸盘数量及分布对柔性件变形的影响可忽略。最后,仿真与实验分析了考虑定位几何量偏差时的加工尺寸偏差,仿真与实验结果规律基本一致,优化装夹后的加工尺寸偏差最大降幅达57.7μm (35%);综上,CFRP柔性件保形加工中变形引起的加工尺寸偏差不容忽略,在“随形-就近”、“定位与真空吸盘组合”原则下优化装夹可以大幅降低变形引起的尺寸偏差。     

Comprehensive solid-phase extraction method for persistent organic pollutants. Validation and application to the analysis of persistent chlorinated pesticides

The Centers for Disease Control and Prevention (CDC) is involved in many epidemiological studies regarding the measurement of chlorinated pesticides and polychlorinated biphenyls in specimens obtained from humans. In addition to these commonly determined analytes, there is a need to include additional persistent organic pollutants (POPs) in our analyses, which further stresses the analyses because sample volumes remain small. Thus, a single method of analysis for all POPs in human serum is needed. CDC has recently developed a semiautomated and comprehensive solid-phase extraction method for POPs. The method is comprehensive since it was optimized for the extraction of many different POP compound classes. We then developed a purification and fractionation scheme that allows (a) separation of different compound classes by particular functionalities and (b) purification of those fractions to remove coextracted interferences. This paper describes the first step in the semiautomated comprehensive extraction and multiple fractionation method developed by CDC for monitoring POPs. In this paper, we validate the analysis of the persistent chlorinated pesticides, a compound class difficult to examine because of their structural diversity, in human plasma. The method was validated against an existing CDC method by using a spiked quality-control serum pool. The concentrations determined for all analytes using both methods were within 2%-14% relative standard deviations. A multilevel (i.e., 3-4 point) matrix spike showed good linearity for the analytes tested (r2 = 0.978-0.999). The method was then applied to 40-year-old archived plasma samples for the quantitative analysis of selected chlorinated pesticides. Mean recoveries of the 13C-labeled internal quantification standards ranged from 64% to 123% for the 11 monitored pesticides. The overall method proved to be robust by handling old coagulated plasma samples. It allowed faster throughput of samples than our previous methods and provided cleaner samples with less frequent interferences or background as analyzed by high-resolution mass spectrometry. The method represents a preliminary step in establishing an automated, comprehensive multiresidue analysis method for POPs in human serum.     

Set-partitioning-based heuristic for balancing and configuration of automated flexible machining line

Flexible machining lines are used in a wide range of industries due to their ability of reconfiguration to meet high variety of customer demands. A novel problem is proposed in the current research to consider automated flexible machining line (AFML) with automated machining using computer numerical control machines and automated auxiliary operations using robots. A mixed-integer programming model for the current novel problem is developed. Moreover, a novel method named set-partitioning-based heuristic (SPH) is proposed to solve this new flexible machining line balancing problem to minimise the cycle time of the line and the performance is compared with both exact algorithm and random search algorithm. A set of benchmark instances based on different size of problems against different system parameters is made. Furthermore, sensitivity analysis of the system parameter in AFML is performed to know, how the number of machines and processing time can influence the cycle time and the utilisation of AFML. Computational experiments are performed to show the performance of the proposed method SPH against other methods and the results indicate that SPH performs best among all test methods in terms of solution quality and computation on both the proposed benchmark instances.     

A cost‐effective approach for rapid manufacturing a low pressure wax injection mold with high surface finish and high dimensional accuracy

Investment casting process is considered as an economic method for mass production of metal parts. Improvement of mold surface quality and geometric accuracy in the fused deposition rapid tooling is a major concern. In this paper, a new technique is proposed for rapid manufacturing a low pressure wax injection mold with high surface finish and high dimensional accuracy. Wax patterns produced from this mold have not only better dimensional accuracy but also better surface finish. The average relative error of dimension of wax patterns can be reduced from 1.76% to 0.66%. Surface roughness improvement rate of wax patterns of up to 85.71% can be achieved. Advantages of this technique include low manufacturing cost, simple manufacturing process and flexible process capability.     

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.     

Nanowire substrate-based laser scanning cytometry for quantitation of circulating tumor cells

We report on the development of a nanowire substrate-enabled laser scanning imaging cytometry for rare cell analysis in order to achieve quantitative, automated, and functional evaluation of circulating tumor cells. Immuno-functionalized nanowire arrays have been demonstrated as a superior material to capture rare cells from heterogeneous cell populations. The laser scanning cytometry method enables large-area, automated quantitation of captured cells and rapid evaluation of functional cellular parameters (e.g., size, shape, and signaling protein) at the single-cell level. This integrated platform was first tested for capture and quantitation of human lung carcinoma cells from a mixture of tumor cells and leukocytes. We further applied it to the analysis of rare tumor cells spiked in fresh human whole blood (several cells per mL) that emulate metastatic cancer patient blood and demonstrated the potential of this technology for analyzing circulating tumor cells in the clinical settings. Using a high-content image analysis algorithm, cellular morphometric parameters and fluorescence intensities can be rapidly quantitated in an automated, unbiased, and standardized manner. Together, this approach enables informative characterization of captured cells in situ and potentially allows for subclassification of circulating tumor cells, a key step toward the identification of true metastasis-initiating cells. Thus, this nanoenabled platform holds great potential for studying the biology of rare tumor cells and for differential diagnosis of cancer progression and metastasis.