An assessment of peptide enrichment methods employing mTRAQ quantification approaches

The human plasma peptidome has potential in biomarker discovery not least because the plasma proteome is a challenging matrix due to its complexity and dynamic range. However, methods to significantly reduce the amount of protein present in plasma while retaining the less abundant peptides present in plasma samples has been a major issue. Here, we present a novel strategy which has been employed to assess the effectiveness of removing interfering proteins while retaining peptides of interest. To monitor peptide retention, a spiked in digested protein, in this case a synthetic QconCAT protein, was employed. This enabled a variety of target analytes (peptides) to be monitored for their retention in liquid phase, providing a broader picture of peptide loss from each method assessed. The incorporation of mTRAQ labeling allowed the presence of each peptide to be monitored, and accurate peptide losses to be determined in a Selected Reaction Monitoring (SRM) assay, thus, enabling an objective semiquantitative conclusion to be drawn regarding the suitability of each method for protein removal and peptide retention. We also assessed a range of methods for retaining nontryptic peptides in a plasma peptidomics workflow. From these data, we determined an optimal workflow for removing intact protein, while retaining peptides for MS-based analyses.     

Universal method for determining electrolyte temperatures in capillary electrophoresis

Temperature increase in capillary electrophoresis (CE) due to Joule heating is an inherent limitation of this powerful separation technique. Active cooling systems can decrease the temperature of a large part of the capillary but they leave "hot spots" at the capillary ends which can completely ruin some CE analyses despite their short lengths. Here, we introduce a "universal method for determining electrolyte temperatures" (UMET) that can determine temperatures in both efficiently- and inefficiently-cooled parts of the capillary. UMET can be applied to all electrolytes, as it does not involve any probe; it requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. To demonstrate the universality of UMET, we measured temperatures for electrolytes of different ionic strengths as well as for different capillary diameters. We further propose a "simplified universal method for predicting electrolyte temperatures" (SUMET) which only requires one measurement of current and voltage (that can be completed in 1 min) and uses two empirical equations to predict temperatures in the efficiently- and inefficiently-cooled parts of the capillary. The equations include several instrument-specific empirical parameters that are determined using a large set of current-voltage data obtained with UMET for a range of electrolytes and different capillaries. To demonstrate the utility of SUMET, we obtained the required data set for a Beckman MDQ CE instrument and produced all required empirical parameters that enable a user of this instrument to predict the temperature for every new experimental set in a matter of minutes. We confirmed the accuracy of SUMET by measuring the temperature-sensitive dissociation rate constant of a protein-DNA complex. We foresee that UMET will be used to produce instrument-specific empirical parameters for all CE instruments and then SUMET will be routinely used for temperature prediction in CE.     

Cooling equilibrium and beam loss with internal targets in high energy storage rings

The beam cooling equilibrium with internal target interaction is analyzed for parameters relevant to the proposed High Energy Storage Ring (HESR). For the proposed experiments with anti-protons high luminosities together with low momentum spreads are required. Rate equations are used to predict the rms equilibrium beam parameters. The cooling and IBS rate coefficients are obtained from simplified models. Energy loss straggling in the target and the associated beam loss are analyzed analytically assuming a thin target. A longitudinal kinetic simulation code is used to study the evolution of the momentum distribution in coasting and bunched beams. Analytic expressions for the target induced momentum tail are found in good agreement with the simulation results.     

Isolation and identification of sialylated glycopeptides from bovine alpha1-acid glycoprotein by off-line capillary electrophoresis MALDI-TOF mass spectrometry

Sialylated glycopeptides contained in liquid chromatographic fractions of bovine alpha1-glycoprotein tryptic digests were isolated from asialo peptides using capillary electrophoresis (CE). CE effluents were deposited directly onto a metallic target and analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This method allowed the characterization of four N-glycosylation sites in the glycoprotein, and each site was observed as a set of sialylated peptide glycoforms. Tandem mass spectrometry was used to confirm peptide sequences and glycan content in glycoforms. The CE method developed for this study resulted in a very clear separation of the sialylated from the asialo content of glycoprotein digests and proved very useful in the determination of the nature and location of sialylated glycans along the protein chain. This article is the first report describing the use of on-target CE fraction collection using a MALDI removable sample concentrator.     

Masonry infilled reinforced concrete frames under horizontal loading / Stahlbetonrahmen mit Ausfachungen aus Mauerwerk unter horizontalen Belastungen

The behaviour of infilled reinforced concrete frames under horizontal load has been widely investigated, both experimentally and numerically. Since experimental tests represent large investments, numerical simulations offer an efficient approach for a more comprehensive analysis. When RC frames with masonry infill walls are subjected to horizontal loading, their behaviour is highly non‐linear after a certain limit, which makes their analysis quite difficult. The non‐linear behaviour results from the complex inelastic material properties of the concrete, infill wall and conditions at the wall‐frame interface. In order to investigate this non‐linear behaviour in detail, a finite element model using a micro modelling approach is developed, which is able to predict the complex non‐linear behaviour resulting from the different materials and their interaction. Concrete and bricks are represented by a non‐linear material model, while each reinforcement bar is represented as an individual part installed in the concrete part and behaving elasto‐plastically. Each brick is modelled individually and connected taking into account the non‐linearity of a brick mortar interface. The same approach is followed using two finite element software packages and the results are compared with the experimental results. The numerical models show a good agreement with the experiments in predicting the overall behaviour, but also very good matching for strength capacity and drift. The results emphasize the quality and the valuable contribution of the numerical models for use in parametric studies, which are needed for the derivation of design recommendations for infilled frame structures.     

Analysis and characterization of switched reluctance motors: Part I /sub y/namic, static, and frequency spectrum analyses

This paper consists of three parts. Section I explores two software applications for modeling and simulating the dynamic characteristics of switched reluctance motors (SRMs). The software applications are the electromechanical structure system (EMSS), which is a tool within the proprietary Maxwell SPICE environment, and power system computer-aided design (PSCAD). The major parameter of an SRM, the nonlinear winding inductance, which determines the dynamic torque, is individually modeled for use with these applications. Simulation of the dynamic torque and the speed for any load is discussed. Section II presents a new stator geometry for SRMs that improves the torque profile. In the new geometry, pole shoes are affixed to the stator poles. The paper discusses a sensitivity study of the torque profile using a two-dimensional finite-element field simulation. Section III presents a frequency spectrum analysis of torque profile using the fast Fourier transform (FFT) capability of MATLAB. Salient features of this analysis for torque profile of SRM and the results of simulation are presented. Section IV concludes the paper.     

A decentralised conflict and error detection and prediction model

The objective of this research is to study the performance of a newly developed decentralised conflict and error detection and prediction model (CEDPM) over different networks. In the CEDPM, conflict and error (CE) detection and prediction agents are deployed at each collaborative unit to detect and predict CEs, and exchange information with the support of a CE detection and prediction protocol. Two metrics, detection and prediction time and conflict severity, are defined to evaluate CEDPM and CE propagation, respectively, for linear, divergence, convergence, and parallel coordination networks in which different task dependences exist among collaborative units. Experiment results show that the CEDPM performs significantly better for networks with parallel activities. The conflict severity reflects network complexity and increases as the dependence between collaborative units increases. The findings are useful for the design of emerging prognostics systems-of-systems.     

运动目标低频矢量宽带谱干涉结构及其应用研究

随着安静型技术的飞速进步,舰船和潜艇的辐射噪声总源级越来越低,但其低频成分很难消除。推导了低频矢量声场双曲线方程形式的声场干涉条纹方程,建立了双曲线参数与目标运动参数的关系。分别利用射线理论和波动理论模拟了低频声场,计算了运动目标声压和水平、垂直振速的干涉谱图,采用短时傅里叶变换与霍夫变换结合的方法对干涉结构图进行处理,估计舰船目标的运动参数。仿真结果表明,利用运动目标的低频矢量宽带干涉谱可以有效估计出目标的运动参数,同时,垂直振速的干涉谱图参数估计结果较前两种谱图误差小。     

Guidelines for designing BJT amplifiers with low 1/f AM and PM noise

In this paper we discuss guidelines for designing linear bipolar junction transistor amplifiers with low 1/f amplitude modulation (AM) and phase modulation (PM) noise. These guidelines are derived from a new theory that relates AM and PM noise to transconductance fluctuations, junction capacitance fluctuations, and circuit architecture. We analyze the noise equations of each process for a common emitter (CE) amplifier and use the results to suggest amplifier designs that minimize the 1/f noise while providing other required attributes such as high gain. Although we use a CE amplifier as an example, the procedure applies to other configurations as well. Experimental noise results for several amplifier configurations are presented.     

Prediction of low-energy collision-induced dissociation spectra of peptides

A kinetic model, based on the "mobile proton" model of peptide fragmentation, was developed to quantitatively simulate the low-energy collision-induced dissociation (CID) spectra of peptides dissociated in a quadrupole ion trap mass spectrometer. The model includes most fragmentation pathways described in the literature, plus some additional pathways based on the author's observations. The model was trained by optimizing parameters within the model for predictions of CID spectra of known peptides. A best set of parameters was optimized to obtain best match between the simulated spectra and the experimental spectra in a training data set. The performance of the mathematical model and the associated optimized parameter set used in the CID spectra simulation was evaluated by generating predictions for a large number of known peptides, which were not included in the training data set. It was shown that the model is able to predict peptide CID spectra with reasonable accuracy in fragment ion intensities for both singly and doubly charged peptide parent ions up to 2000 u in mass. The optimized parameter set was evaluated to gain insight into the collision-induced peptide fragmentation process.     

Predicting retention time shifts associated with variation of the gradient slope in peptide RP-HPLC

We have developed a sequence-specific model for predicting slopes (S) in the fundamental equation of linear solvent strength theory for the reversed-phase HPLC separation of tryptic peptides detected in a typical bottom-up-proteomics experiment. These slopes control the variation in the separation selectivity observed when the physical parameters of chromatographic separation, such as gradient slope, flow rate, and column size are altered. For example, with the use of an arbitrarily chosen set of tryptic peptides with a 4-times difference in the gradient slope between two experiments, the R(2)-value of correlation between the observed retention times of identical species decreases to ~0.993-0.996 (compared to a theoretical value of ~1.00). The observed retention time shifts associated with variations of the gradient slope can be predicted a priori using the approach described here. The proposed model is based on our findings for a set of synthetic species (Vu, H.; Spicer, V.; Gotfrid, A.; Krokhin, O. V. J. Chromatogr., A, 2010, 1217, 489-497), which postulate that slopes S can be predicted taking into account simultaneously peptide length, charge, and hydrophobicity. Here we extend this approach using an extensive set of real tryptic peptides. We developed the procedure to accurately measure S-values in nano-RP HPLC MS experiments and introduced sequence-specific corrections for a more accurate prediction of the slopes S. A correlation of ~0.95 R(2)-value between the predicted and experimental S-values was demonstrated. Predicting S-values and calculating the expected retention time shifts when the physical parameters of separation like gradient slope are altered will facilitate a more accurate application of peptide retention prediction protocols, aid in the transfer of scheduled MRM (SRM) procedures between LC systems, and increase the efficiency of interlaboratory data collection and comparison.     

Comparison of linear and nonlinear analysis in estimating the Thomas model parameters for methylene blue adsorption onto natural zeolite in fixed-bed column

Comparison analysis of linear least square method and nonlinear least square method for estimating the kinetic parameters was made using the experimental column data of methylene blue (MB) adsorption onto zeolite at different flow rates and initial concentration. The data were fitted to Thomas model equations using linear and nonlinear regressive analysis, respectively. The error analysis was performed. Present investigation showed that the linear and nonlinear methods are both suitable to predict the breakthrough curves using Thomas model parameters and the nonlinear method is better.     

Development of a NIST standard reference material containing thirty volatile organic compounds at 5 nmol/mol in nitrogen

Primary gravimetric gas cylinder standards containing 30 volatile organic compounds (VOCs) in nitrogen were prepared using a procedure previously developed to prepare gas mixture cylinder standards of VOCs at the 5 nmol/mol level. This set of primary standards was intercompared to existing gas cylinder standards, containing as many as 19 of the 30 volatile organics present in these new primaries, using gas chromatography with a hydrogen flame ionization detector coupled with cryogenic preconcentration. The linear regression analysis showed excellent agreement among the standards for each compound. Similar mixtures containing many of these compounds in treated aluminum gas cylinders have been evaluated over time and have shown stability for as much as 10 years. The development of these 30-component primary standards led to the preparation and certification of a reissue of Standard Reference Material (SRM) 1804 at the nominal amount-of-substance fraction of 5 nmol/mol for each analyte. A lot of 20 cylinders containing the mixture was prepared at NIST following previously demonstrated protocols for preparation of the cylinders. Each cylinder was analyzed against one cylinder from the lot, designated as the "lot standard," for each of the 30 compounds. As a result of the uncertainty analysis, the data showed that rather than declaring the lot homogeneous with a much higher uncertainty, each cylinder could be individually certified. The expanded uncertainty limits ranged from 1.5 to 10% for 28 of the 30 analytes, with two of the analytes having uncertainties as high as 19% in those SRM cylinders certified. Due to stability issues and some high uncertainties for a few analytes in 2 of the samples, 18 of the 20 candidate SRM samples were certified. These volatile organic gas mixtures represent the most complex gas SRMs developed at NIST.     

Computing Transition Probability in Markov Chain for Early Prediction of Software Reliability

Early prediction of software reliability provides basis for evaluating potential reliability during early stages of a project. It also assists in evaluating the feasibility of proposed reliability requirements and provides a rational basis for design and allocation decisions. Many researchers have proposed different approaches to predict the software reliability based on a Markov model. The transition probabilities in between the states of the Markov model are input parameters to predict the software reliability. In the existing approaches, these probabilities are either assumed on some knowledge or computed using analytical method, and hence, it does not give accurate predicted reliability figure. Some authors compute them using operational profile data, but that is possible only after the deployment of the software, and this is not early prediction. The work in this paper is devoted to demonstrate the computation of transition probability in the Markov reliability model taking a case study. The proposed approach has been validated on 47 sets of real data. Copyright © 2015 John Wiley & Sons, Ltd.     

基于Witness的看板生产系统仿真与参数优化研究

以看板生产系统基本理论为基础,某发动机总装车间为实证案例,应用Witness物流仿真软件对该车间看板生产系统进行仿真与优化,实现在短时间内预见实施效果,对系统绩效如设备利用率、订单处理效率等做出评价。结果表明,实施看板系统后,订单得到及时响应,提高了客户满意度,设备平均利用率和生产效率显著提高,产品产量达到目标产量。最后对看板生产系统的隐性参数进行优化,为不同企业根据实际生产情况实施看板生产系统参数选取提供依据     

基于改进Ibarra-Medina-Krawinkler材料模型的RC圆柱恢复力模型参数识别

该文基于美国太平洋地震工程研究中心结构性能数据库中的76根弯曲破坏及弯剪破坏的钢筋混凝土(Reinforced concrete, RC)圆柱试验数据,以峰值指向型改进Ibarra-Medina-Krawinkler (I-M-K)材料模型作为恢复力模型,采用OpenSEES有限元平台对所选择的RC圆柱试验数据进行数值模拟,识别得到了每一RC圆柱的恢复力模型参数。选取RC圆柱的15个基本设计参数为基本变量,采用多元线性回归和分步拟合,获得了RC圆柱恢复力模型参数的经验预测公式(值)。通过对试验验证组中3根RC圆柱的滞回试验数据进行模拟,对该文提出的RC圆柱恢复力模型参数的经验预测公式(值)的合理性进行了验证。结果表明:该文获得的经验预测公式(值)可以较为准确地预测RC圆柱的恢复力模型参数,可以较合理地反映RC圆柱在循环加载过程中的强度及刚度的退化特征。     

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.     

On-line capillary electrophoresis/microelectrospray ionization-tandem mass spectrometry using an ion trap storage/time-of-flight mass spectrometer with SWIFT technology.

The development of a system capable of the speed required for on-line capillary electrophoresis-tandem mass spectrometry (CE-MS/MS) of tryptic digests is described. The ion trap storage/reflectron time-of-flight (IT/reTOF) mass spectrometer is used as a nonscanning detector for rapid CE separation, where the peptides are ionized on-line using electrospray ionization (ESI). The ESI produced ions are stored in the ion trap and dc pulse injected into the reTOF-MS at a rate sufficient to maintain the separation achieved by CE. Using methodology generated by software and hardware developed in our lab, we can produce SWIFT (Stored Waveform Inverse Fourier Transform) ion isolation and TICKLE activation/fragmentation voltage waveforms to generate MS/MS at a rate as high as 10 Hz so that the MS/MS spectra can be optimized on even a 1-2 s eluting peak. In CE separations performed on tryptic digests of dogfish myelin basic protein (MBP) where eluting peaks 4-8 s wide are observed, it is demonstrated that an acquisition rate of 4 Hz provides > 20 spectra/peak and is more than sufficient to provide optimized MS/MS spectra of each of the eluting peaks in the electropherogram. The detailed structural analysis of dogfish MBP including several posttranslational modifications using CE-MS and CE-MS/MS is demonstrated using this method with < 10 fmol of material consumed.     

Nonaqueous electrophoresis microchip separations: conductivity detection in UV-absorbing solvents

The use of organic solvents in microfabricated capillary electrophoresis (CE) devices is demonstrated in connection with the separation of aliphatic amines in pure dimethylformamide, dimethylacetamide, dimethyl sulfoxide, or propylene carbonate media. Contactless conductivity detection is employed for monitoring the separated solutes in these UV-absorbing solvents. The effect of the physicochemical properties of the organic solvent upon the migration behavior is investigated. The apparent mobility increases nearly linearly with the reciprocal of the solvent viscosity, while the electroosmotic mobility increases in a linear fashion with the dielectric constant/ viscosity ratio. Some deviation from theoretical predictions is observed using propylene carbonate. The nonaqueous CE microchip offers high separation efficiency, reflected in plate numbers ranging from 93,680 to 127,680, using a separation voltage of +3,000 V, a dimethylformamide medium, and a contactless conductivity detection. Experimental parameters affecting the analytical performance of the nonaqueous CE/conductivity microchip are examined. Calibration and precision experiments indicate a linear and reproducible response. Such use of organic solvents can benefit microchip separations through extended scope (toward nonpolar solutes) and tunable selectivity.