Silver- and Coomassie-staining protocols: detection limits and compatibility with ESI MS

Staining protocols for PAGE have to be sensitive and should not impair further MS analysis of selected samples. In this study, the MS compatibility of different silver- and Coomassie-staining protocols with a nano-LC-MS/MS system was systematically elucidated. Altogether, 13 different silver-staining, 1 imidazole-staining and 2 Coomassie-staining protocols were used and compared to each other for their achieved sequence coverage and their detection sensitivity. Three proteins were used as model proteins (bovine serum albumin, rabbit L-lactate dehydrogenase, bovine milk beta-lactoglobulin) in decreasing concentration (12 pmol down to 30 fmol) and different staining protocols were applied. The conclusion of this study is that two silver-staining protocols (Blum, H. et al.,. Electrophoresis 1987, 8, 93-99 and Shevchenko, A. et al.,. Anal. Chem. 1996, 68, 850-858) combine good sequence coverage and good sensitivity and are recommended for nano-LC-MS/MS analysis.     

Ab initio rotation-vibration spectra of HCN and HNC

We have calculated an ab initio HCN/HNC linelist for all transitions up to J= 25 and 18000 cm(-1) above the zero point energy. This linelist contains more than 200 million lines each with frequencies and transition dipoles. The linelist has been calculated using our semi-global HCN/HNC VQZANO + PES and dipole moment surface, which were reported in van Mourik et al. (J. Chem. Phys. 115 (2001) 3706). With this linelist we synthesise absorption spectra of HCN and HNC at 298 K and we present the band centre and band transition dipoles for the bands which are major features in these spectra. Several of the HCN bands and many of the HNC bands have not been previously studied. Our line intensities reproduce via fully ab initio methods the unusual intensity structure of the HCN CN stretch fundamental (00(0)1) for the first time and also the forbidden (02(2)0) HCN bending overtone. We also compare the J = 1-->0 pure rotational transition dipole in the HCN/HNC ground and vibrationally excited states with experimental and existing ab initio results.     

Use of engineered unique cysteine residues to facilitate oriented coupling of proteins directly to a gold substrate

A prerequisite for any "lab on a chip" device that utilizes an electrical signal from the sensor protein is the ability to attach the protein in a specific orientation onto a conducting substrate. Here, we demonstrate the covalent attachment to a gold surface of light-harvesting membrane proteins, from Rhodobacter sphaeroides, via cysteine (Cys) residues engineered on either the cytoplasmic or periplasmic face. This simple directed attachment is superior in its ability to retain light-harvesting complex (LHC) function, when compared to a similar attachment procedure utilizing a self-assembled monolayer on gold. LH 1 has previously been observed to have superior photostability over LH 2 (Magis et al. [2010] Biochim. Biophys. Acta, 1798, 637-645); this characteristic is maintained even with the introduction of Cys residues.     

A dual-level state-specific time-dependent density-functional theory

A highly efficient new algorithm for time-dependent density-functional theory (TDDFT) calculations is presented. In this algorithm, a dual-level approach to speed up DFT calculations (Nakajima and Hirao, J Chem Phys 2006, 124, 184108) is combined with a state-specific (SS) algorithm for TDDFT (Chiba et al., Chem Phys Lett 2006, 420, 391). The dual-level SS-TDDFT algorithm was applied to excitation energy calculations of typical small molecules, the Q bands of the chlorophyll A molecule, the charge-transfer energy of the zincbacteriochlorin-bacteriochlorin model system, and the lowest-lying excitation of the circumcoronene molecule. As a result, it was found that the dual-level SS-TDDFT gave correct excitation energies with errors of 0.2-0.3 eV from the standard TDDFT approach, with much lower CPU times for various types of excitation energies of large-scale molecules.     

Simulation of a crossover from the precipitation wave to moving Liesegang pattern formation

Model simulations to investigate the precipitation wave phenomenon and a crossover from the precipitation wave to moving Liesegang patterns were performed. The chemical scheme contains four chemical species via the interaction of precipitation and redissolution (complex formation), in which the precipitation reaction term was based on Ostwald's supersaturation theory. In this article, for the first time, all the features and behaviors of the heterogeneous traveling waves are reproduced, which were observed experimentally in the work of Zrínyi et al. (Zrínyi, et al. J. Phys. Chem. 1991, 95, 1618.). The detailed investigation of the pattern formation showed three possible states of the system, which depend on the initial concentration of the inner and outer electrolytes, respectively. These are the precipitation wave (single moving precipitation band), the moving Liesegang pattern (moving precipitation bands), and the state where these two patterns coexist.     

Comments on the article "On evaluating molecular-docking methods for pose prediction and enrichment factors"

The recent article "On Evaluating Molecular-Docking Methods for Pose Prediction and Enrichment Factors" (Chen H. et al. J. Chem. Inf. Model. 2006, 46, 401-415) contains a series of comments on a similar study we published in Proteins in 2004 (Perola et al. Proteins 2004, 56, 235-249). We believe that some of these comments are misleading, and we feel that an adequate response is in order.     

Parallel processing in the isoelectric focusing chip

Investigation of isoelectric focusing (IEF) kinetics has been performed to provide the theoretical basis for miniaturization of classical IEF in immobilized pH-gradients. Standard IEF demands colinearity of the electric field and pH-gradient directions (serial devices). It is shown that the IEF separation process based on a continuous, serial pH gradient is incompatible with miniaturization of separation devices. The new realization of the IEF device by a parallel IEF chip is suggested and analyzed. The main separation tool of the device is a dielectric membrane (chip) with conducting channels that are filled by Immobiline gels of varying pH. The membrane is held perpendicular to the applied electric field and proteins are collected (trapped) in the channels whose pH are equal to the pI of the proteins. The pH value of the surrounded aqueous solution is not equal to any channel's pH. The fast particle transport between different channels takes place due to convection in the aqueous solution. The new device geometry introduces two new spatial scales to be considered: the scale of transition region from a solution to the gel in a channel and a typical channel size. The corresponding time scales defining the IEF process kinetics are analyzed and scaling laws are obtained. It is shown both theoretically and experimentally that parallel IEF accelerates the fractionation of proteins by their pI down to several minutes and enables possible efficient sample collection and purification.     

Overlapping resonances in the control of intramolecular vibrational redistribution

Coherent control of bound state processes via the interfering overlapping resonance scenario [Christopher et al., J. Chem. Phys. 123, 064313 (2006)] is developed to control intramolecular vibrational redistribution (IVR). The approach is applied to the flow of population between bonds in a model of chaotic OCS vibrational dynamics, showing the ability to significantly alter the extent and rate of IVR by varying quantum interference contributions.     

Fabrication of micro free-flow electrophoresis chip by photocurable monomer binding microfabrication technique for continuous separation of proteins and their numerical simulation

In this study, a simple, fast, and reliable method to fabricate a micro free-flow electrophoresis (μFFE) device on glass is presented. The two-dimensional depth channel in the chip was easily achieved by using a photocurable monomer (NOA?81) that served as the bonding material. In such a geometrical structure (two-dimensional depth channel), the effect of fluid behavior on the separation efficiency of micro free-flow zone electrophoresis (μFFZE) was simulated. The results of numerical simulation indicate that the pressure at the inlets may play an important role in the separation performance. Under the optimum separation conditions, four FITC-labeled amino acids were well separated, indicating the validity of the performance of the chip. Since the chip was fabricated by organic polymer bonding, it was easily recyclable through a simple re-fabrication process. The reproducibility of results from these recycling re-fabrication chips was investigated. The RSD of the resolution between FITC-l-glycine and FITC-l-phenylalanine was 5.3%. Furthermore, three FITC-labeled proteins were successfully separated with the resolution of 2.2 and 5.46, respectively, by using the coating of neutral liposome.     

Microfluidic distillation chip for methanol concentration detection

An integrated microfluidic distillation system is proposed for separating a mixed ethanol-methanol-water solution into its constituent components. The microfluidic chip is fabricated using a CO₂ laser system and comprises a serpentine channel, a boiling zone, a heating zone, and a cooled collection chamber filled with de-ionized (DI) water. In the proposed device, the ethanol-methanol-water solution is injected into the microfluidic chip and driven through the serpentine channel and into the collection chamber by means of a nitrogen carrier gas. Following the distillation process, the ethanol-methanol vapor flows into the collection chamber and condenses into the DI water. The resulting solution is removed from the collection tank and reacted with a mixed indicator. Finally, the methanol concentration is inversely derived from the absorbance measurements obtained using a spectrophotometer. The experimental results show the proposed microfluidic system achieves an average methanol distillation efficiency of 97%. The practicality of the proposed device is demonstrated by detecting the methanol concentrations of two commercial fruit wines. It is shown that the measured concentration values deviate by no more than 3% from those obtained using a conventional bench top system.     

Implementation of empirical dispersion corrections to density functional theory for periodic systems

A recently developed empirical dispersion correction (Grimme et al., J. Chem. Phys. 2010, 132, 154104) to standard density functional theory (DFT‐D3) is implemented in the plane‐wave program package VASP. The DFT‐D3 implementation is compared with an implementation of the earlier DFT‐D2 version (Grimme, J. Comput. Chem. 2004, 25, 1463; Grimme, J. Comput. Chem. 2006, 27, 1787). Summation of empirical pair potential terms is performed over all atom pairs in the reference cell and over atoms in shells of neighboring cells until convergence of the dispersion energy is obtained. For DFT‐D3, the definition of coordination numbers has to be modified with respect to the molecular version to ensure convergence. The effect of three‐center terms as implemented in the original molecular DFT‐D3 version is investigated. The empirical parameters are taken from the original DFT‐D3 version where they had been optimized for a reference set of small molecules. As the coordination numbers of atoms in bulk and surfaces are much larger than in the reference compounds, this effect has to be discussed. The results of test calculations for bulk properties of metals, metal oxides, benzene, and graphite indicate that the original parameters are also suitable for solid‐state systems. In particular, the interlayer distance in bulk graphite and lattice constants of molecular crystals is considerably improved over standard functionals. With the molecular standard parameters (Grimme et al., J. Chem. Phys. 2010, 132, 154104; Grimme, J. Comput. Chem. 2006, 27, 1787) a slight overbinding is observed for ionic oxides where dispersion should not contribute to the bond. For simple adsorbate systems, such as Xe atoms and benzene on Ag(111), the DFT‐D implementations reproduce experimental results with a similar accuracy as more sophisticated approaches based on perturbation theory (Rohlfing and Bredow, Phys. Rev. Lett. 2008, 101, 266106). © 2012 Wiley Periodicals, Inc.     

Time-sliced velocity-mapped imaging studies of the predissociation of single ro-vibronic energy levels of N2 in the extreme ultraviolet region using vacuum ultraviolet photoionization

The predissociation of N(2) from the rotational levels in the o(1)∏(u) (v(') = 2) and b(') (1)Σ(u) (v(') = 8) bands has been studied in the wavenumber (or energy) range from 109?350 cm(-1) (13.5577 eV) to 109?580 cm(-1) (13.5862 eV) by time-sliced velocity-mapped imaging technique with VUV photoionization detection of the fragments. These levels were excited from the ground state of N(2) (X(1)Σ(g) (+), v(") = 0) levels using an unfocused vacuum ultraviolet (VUV) laser via a one-photon process. The same VUV laser is used to ionize the metastable N ((2)D(o)) produced from the predissociation process and the time-sliced velocity-mapped imaging technique is used to determine their velocity and angular distributions. Two different theoretical methods developed, respectively, by Kim et al. [J. Chem. Phys. 125, 133316 (2006) and Zande [J. Chem. Phys. 107, 9447 (1997)] were used to calculate the anisotropic parameters for the predissociation to the channel N((4)S(o)) + N((2)D(o)) to compare with the observed value for each of the rotational levels. Very good agreement with the experimental results was obtained for both methods. Possible predissociation mechanisms were predicted from the measurements and calculations.     

Performing microchannel temperature cycling reactions using reciprocating reagent shuttling along a radial temperature gradient

This study develops a novel temperature cycling strategy for executing temperature cycling reactions in laser-etched poly(methylmethacrylate) (PMMA) microfluidic chips. The developed microfluidic chip is circular in shape and is clamped in contact with a circular ITO heater chip of an equivalent diameter. Both chips are fabricated using an economic and versatile laser scribing process. Using this arrangement, a self-sustained radial temperature gradient is generated within the microfluidic chip without the need to thermally isolate the different temperature zones. This study demonstrates the temperature cycling capabilities of the reported microfluidic device by a polymerase chain reaction (PCR) process using ribulose 1,5-bisphosphate carboxylase large subunit (rbcL) gene as a template. The temperature ramping rate of the sample inside the microchannel is determined from the spectral change of a thermochromic liquid crystal (TLC) solution pumped into the channel. The present results confirm that a rapid thermal cycling effect is achieved despite the low thermal conductivity of the PMMA substrate. Using IR thermometry, it is found that the radial temperature gradient of the chip is approximately 2 degrees C mm(-1). The simple system presented in this study has considerable potential for miniaturizing complex integrated reactions requiring different cycling parameters.     

Distillation and detection of SO2 using a microfluidic chip

A miniaturized distillation system is presented for separating sulfurous acid (H(2)SO(3)) into sulfur dioxide (SO(2)) and water (H(2)O). The major components of the proposed system include a microfluidic distillation chip, a power control module, and a carrier gas pressure control module. The microfluidic chip is patterned using a commercial CO(2) laser and comprises a serpentine channel, a heating zone, a buffer zone, a cooling zone, and a collection tank. In the proposed device, the H(2)SO(3) solution is injected into the microfluidic chip and is separated into SO(2) and H(2)O via an appropriate control of the distillation time and temperature. The gaseous SO(2) is then transported into the collection chamber by the carrier gas and is mixed with DI water. Finally, the SO(2) concentration is deduced from the absorbance measurements obtained using a spectrophotometer. The experimental results show that a correlation coefficient of R(2) = 0.9981 and a distillation efficiency as high as 94.6% are obtained for H(2)SO(3) solutions with SO(2) concentrations in the range of 100-500 ppm. The SO(2) concentrations of two commercial red wines are successfully detected using the developed device. Overall, the results presented in this study show that the proposed system provides a compact and reliable tool for SO(2) concentration measurement purposes.     

Selective extraction of size-fractioned DNA samples in microfabricated electrophoresis devices

We have designed and constructed a microfabricated device for separation of double-stranded DNA fragments using a crosslinked sieving medium and spatially selective extraction of the desired fraction. Based on measuring the width and spacing of migrating bands, a narrow side channel is constructed perpendicular to the separation channel to collect the DNA fragments of interest. This selective collection technique was tested using a 100 base pair double-stranded DNA ladder. We successfully demonstrate selective extraction of the desired fragment with minimal interference from the adjacent bands in an electric field of 31 V/cm. We also achieve extraction of multiple DNA fragments using an array of microelectrodes in this side channel. The device uses cross-linked polyacrylamide gel matrix, allowing the separation to be performed in a distance of 1 cm or less and at a low electric field strength. Together with on-chip electrode, this design is amenable to integration with reaction chambers into a single device for portable genetic-based analysis.     

Projection of two-dimensional diffusion in a curved midline and narrow varying width channel onto the longitudinal dimension

This study focuses on the derivation of a general effective diffusion coefficient to describe the two-dimensional (2D) diffusion in a narrow and smoothly asymmetric channel of varying width, in the simple diffusional motion of noninteracting pointlike particles under no external field. We present a generalization to the case of an asymmetric channel using the projection method introduced earlier by Kalinay and Percus [J. Chem. Phys. 122, 204701 (2005); and Phys. Rev. E 74, 041203 (2006)] to project the 2D diffusion equation into an effective one-dimensional generalized Fick-Jacobs equation. The expression for the diffusion coefficient given in Eq. (23) is our main result. This expression is a more general effective diffusion coefficient for narrow channels in 2D, which contains the well-known previous results as special cases, namely, those obtained by Bradley [Phys. Rev. E 80, 061142 (2009)], and more recently by Berezhkovskii and Szabo [J. Chem. Phys. 135, 074108 (2011)]. Finally, we study some specific 2D asymmetric channel configurations to test and show the broader applicability of Eq. (23).     

Interface solution isoelectric focusing with in situ MALDI‐TOF mass spectrometry

This paper describes a simple and reusable microfluidic device combining solution IEF (sIEF) with MALDI‐TOF MS for rapid proteomic and metabolic analysis of microliter samples. The device contains two glass plates with nanoliter microwell arrays, which can be assembled to form a fluidic path for sIEF separation, and reconfigured for dividing separated bands. One microliter samples can be loaded and separated by sIEF into static bands in 10～30 min. After a slipping operation, the static IEF bands can be divided into nanoliter droplets in microwells without mobilization, and the device can be opened for in situ MALDI‐TOF MS detection without loss of separation resolution. The performance of the device is characterized by separating and identifying intact proteins. The applicability in metabolic analysis is demonstrated by preliminary experiments on profiling small molecular metabolites in cerebrospinal fluid microdialysates from rat brain.     

Rapid and sensitive separation of trace level protein digests using microfabricated devices coupled to a quadrupole--time-of-flight mass spectrometer

The application of microfabricated devices coupled to a quadrupole time-of-flight mass spectrometer (Qq-TOF-MS) is presented for the analysis of trace level digests of gel-isolated proteins. In order to enhance the sample loading for proteomics analyses, two different on-chip sample preconcentration techniques were evaluated. First, a sample stacking procedure that used polarity switching to remove the sample buffer prior to zone electrophoresis was easily integrated on the microfabricated devices. With the present chip design, this preconcentration technique provided up to 70 nL sample injection with sub-nM detection limits for most peptide standards. For applications requiring larger sample loading, a disposable adsorption preconcentrator using a C18 membrane is incorporated outside the chip. This preconcentration method yielded lower peptide recoveries than that obtainable with sample stacking, and provided a convenient means of injecting several microL of sample with detection limits of typically 2.5 nM for hydrophobic peptides. The analytical merits of both sample enrichment approaches are described for the identification of bands isolated from two-dimensional (2-D) gel separation of protein extracts from Haemophilus influenzae. Accurate molecular mass measurements (< 5 ppm) in peptide mapping experiments is obtained by introducing an internal standard via a post-separation channel. Rapid identification of trace level peptides is also demonstrated using on-line tandem mass spectrometry and database searching with peptide sequence tags.     

Monolithic column plastic microfluidic device for peptide analysis using electrospray from a channel opening on the edge of the device

Electrospray from a channel exit at the edge of a fluorocarbon coated cycloolefin copolymer microfluidic device has been investigated. The fluorocarbon coating facilitated generation of a stable electrospray, thereby enhancing the detectability of electrospray ionization (ESI) mass spectrometry (MS). A microfluidic device of integrated ESI emitters and monolithic liquid chromatography columns has been fabricated on a cycloolefin copolymer chip. The monolithic columns were polymerized in situ using UV irradiation with a photomask to confine the porous polymer monolith to the desired regions of the channels. The monolithic stationary phase was homogenous and well bonded to the channel surfaces, which had been functionalized by graft polymerization. The ESI potential was applied within the channel via a carbon ink line. The performance of this microfluidic device was demonstrated by analysis of a tryptic digest of bovine serum albumin on an ion trap MS instrument.     

An accurate and efficient empirical approach for calculating the dielectric self-energy and ion-ion pair potential in continuum models of biological ion channels

This paper presents empirical formulas for calculating the dielectric self-energy and ion-ion pair interactions in cylindrical ion channels. The proposed approach can be extended to more complex channel structures, for example, (i) a "straight" channel with variable radius and (ii) a "curved" channel with constant radius. For calibration purposes, we compare results obtained based on the approximate effective potentials developed herein to exact electrostatic calculations obtained via the algorithm of Graf et al.: the agreement is satisfactory. A dynamic lattice Monte Carlo (DLMC) technique is used to further assess the accuracy and efficiency of the proposed empirical potentials. The concentration profiles and current-voltage curves produced with our simple empirical energy formulas are in excellent agreement with numerical results obtained using the algorithm of Graf et al., which calculates all relevant electrostatic forces exactly. The use of effective ion-ion potentials greatly reduces the computer memory required to perform DLMC ion permeation simulations in dielectrically inhomogeneous environments, thus enabling treatment of larger systems than can be handled by numerically exact techniques.