Concentration of DNA in a flowing stream for high-sensitivity capillary electrophoresis

A novel sample pretreatment device is described, and its application to the concentration and purification of crude DNA samples in a flowing stream for subsequent capillary electrophoresis is demonstrated. The device consists of two gap junctions, each covered with a conductive membrane material and built upon a flow channel made of PEEK tubing. Upon the application of an electric field between the junctions, the negatively charged DNA fragments can resist the hydrodynamic flow stream and are trapped between the junctions. DNA fragments dissolved in microliter volumes are captured in a nanoliter-sized band by simply pushing the sample solution through the device. Depending on their electrophoretic mobility, other interfering materials in a crude sample can be removed from the trapped DNA fragments by washing. The selective permeability of the membrane to small ions allows efficient desalting. The concentrated and purified DNA fragments are released by simply turning off or reversing the electric field. Recovery is up to 95%. Performance of the device was evaluated using crude products of fluorescent dye-primer cycle-sequencing reactions. Compared to these crude reaction products, samples purified in the capture device and subsequently collected showed dramatically enhanced signal and resolution when run on a conventional capillary-electrophoresis instrument. Furthermore, the device could be connected in-line to a capillary system for direct injection. The device has great potential for enabling lab-on-a-chip systems to be used with real-world samples.     

A microfluidic electrocapture device in sample preparation for protein analysis by MALDI mass spectrometry

The design and operation of a microfluidic device for sample preparation in MALDI mass spectrometry of peptides and proteins is described. It is particularly useful for proteomics applications and for mass determination of proteins in salt- and detergent-containing solutions. The system consists of a flow channel with two conductive areas or electrical junctions where proteins and peptides are retained by means of an electric field. The microfluidic device is made of PEEK tubing, and the junctions are covered with a conductive polymeric membrane. A syringe pump connected to the device produces a flow stream, and injection of sample is carried out manually via hydrodynamic pressure. Proteolytic peptides and intact proteins in salt- and detergent-containing acidic media were captured at the cathode junction followed by exchange of the original solution to a solvent suitable for subsequent mass spectrometry. Using this principle, a significant desalting effect was obtained for tryptic peptides in mass-mapping experiments. Protein sequence coverages were high (up to 40%) at subpicomole levels with results better than those obtained using reversed-phase solid-phase extraction. In contrast to the latter technique, the microfluidic device has the capacity to efficiently remove detergents such as CHAPS before peptide mapping and protein analysis.     

A device for automated direct sampling and quantitation from solid-phase sorbent extraction cards by electrospray tandem mass spectrometry

A new solid-phase extraction (SPE) device in the 96-well format (SPE Card) has been employed for automated off-line sample preparation of low-volume urine samples. On-line automated analyte elution via SPE and direct quantitation by micro ion spray mass spectrometry is reported. This sample preparation device has the format of a microtiter plate and is molded in a plastic frame which houses 96 separate sandwiched 3M Empore sorbents (0.5-mm-thickness, 8-microm particles) covered on both sides by a microfiber support material. Ninety-six discrete SPE zones, each 7 mm in diameter, are imbedded into the sheet in the conventional 9-mm pitch (spacing) of a 96-well microtiter plate. In this study one-quarter of an SPE Card (24 individual zones) was used merely as a convenience. After automated off-line interference elution of applied human urine from 24 samples, a section of SPE Card is mounted vertically on a computer-controlled X, Y, Z positioner in front of a micro ion spray direct sampling tube equipped with a beveled tip. The beveled tip of this needle robotically penetrates each SPE elution zone (sorbent disk) or stationary phase in a serial fashion. The eluted analytes are sequentially transferred directly to a microelectrosprayer to obtain tandem mass spectrometric (MS/MS) analysis. This strategy precludes any HPLC separation and the associated method development. The quantitative determination of Ritalin (methylphenidate) from fortified human urine samples is demonstrated. A trideuterated internal standard of methylphenidate was used to obtain ion current response ratios between the parent drug and the internal standard. Human control urine samples fortified from 6.6 to 3300 ng/mL (normal therapeutic levels have been determined in other studies to be between 50 and 100 ng/mL urine) were analyzed and a linear calibration curve was obtained with a correlation coefficient of 0.9999, where the precision of the quality control (QC) samples ranged from 9.6% at the 24 ng/mL QC level to 1.2% at the 3000 ng/mL QC level, and the accuracy for the four levels of QC samples ranged from 98.1% to 100.3%. The QC samples were prepared at four concentrations which included 24, 240, 1200, and 3000 ng/mL, respectively. The run time per sample in this work was 1.5 min not including the sample preparation time.     

Forensic identification of neat ricin and of ricin from crude castor bean extracts by mass spectrometry

The protein toxin ricin, which originates from the seeds of Ricinus communis plants, has been the subject of increased interest, due to its potential terrorist use. Exceptionally, this toxin is also subject to the Chemical Weapons Convention. In this paper, it is shown that mass spectrometry can be used to unambiguously verify the presence of ricin in crude toxin preparations. It is demonstrated that MALDI MS can be used for screening, either by direct analysis or by trypsin digestion and peptide mapping. Purified ricin from several varieties of R. communis was characterized by LC-ES MS(/MS). A crude ricin preparation from a single bean was similarly characterized. An LC method was set up with product ion MS/MS detection of selected marker peptides specific for ricin: T5, T7, T11, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. This method was then used to unambiguously identify ricin in a crude preparation of ricin. The MALDI MS molecular weight analysis and the marker peptides LC-ES MS/MS analysis give a forensic level of identification of ricin when combined with activity testing.     

Improved precision of iTRAQ and TMT quantification by an axial extraction field in an Orbitrap HCD cell

Improving analytical precision is a major goal in quantitative differential proteomics as high precision ensures low numbers of outliers, a source of false positives with regard to quantification. In addition, higher precision increases statistical power, i.e., the probability to detect significant differences. With chemical labeling using isobaric tags for relative and absolute quantitation (iTRAQ) or tandem mass tag (TMT) reagents, quantification is based on the extraction of reporter ions from tandem mass spectrometry (MS/MS) spectra. We compared the performance of two versions of the LTQ Orbitrap higher energy collisional dissociation (HCD) cell with and without an axial electric field with regard to reporter ion quantification. The HCD cell with the axial electric field was designed to push fragment ions into the C-trap and this version is mounted in current Orbitrap XL ETD and Orbitrap Velos instruments. Our goal was to evaluate whether the purported improvement in ion transmission had a measurable impact on the precision of MS/MS based quantification using peptide labeling with isobaric tags. We show that the axial electric field led to an increased percentage of HCD spectra in which the complete set of reporter ions was detected and, even more important, to a reduction in overall variance, i.e., improved analytical precision of the acquired data. Notably, adequate precision of HCD-based quantification was maintained even for low precursor ion intensities of a complex biological sample. These findings may help researchers in their design of quantitative proteomics studies using isobaric tags and establish HCD-based quantification on the LTQ Orbitrap as a highly precise approach in quantitative proteomics.     

Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins

Phosphopeptides are often detected with low efficiency by MALDI MS analysis of peptide mixtures. In an effort to improve the phosphopeptide ion response in MALDI MS, we investigated the effects of adding low concentrations of organic and inorganic acids during peptide sample preparation in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. Phosphoric acid in combination with 2,5-DHB matrix significantly enhanced phosphopeptide ion signals in MALDI mass spectra of crude peptide mixtures derived from the phosphorylated proteins alpha-casein and beta-casein. The beneficial effects of adding up to 1% phosphoric acid to 2,5-DHB were also observed in LC-MALDI-MS analysis of tryptic phosphopeptides of B. subtilis PrkC phosphoprotein. Finally, the mass resolution of MALDI mass spectra of intact proteins was significantly improved by using phosphoric acid in 2,5-DHB matrix.     

Continuous-flow pI-based sorting of proteins and peptides in a microfluidic chip using diffusion potential

Efficient sample preparation tools are the key to measuring molecular signals in a complex biological system. A novel continuous-flow isoelectric point (pI)-based sorting technique has been developed for proteins and peptides in a microfluidic chip format. It can sort biomolecules at a relatively high flow rate of up to 10 microL/min and does not require carrier ampholytes, which can create molecular backgrounds for subsequent analysis. Furthermore, the electrophoretic field required to run the pI-based sorting is generated by the diffusion of buffer ions in situ, at the liquid junction between two laminar flows within the microfluidic channel. Utilizing the diffusion potential in combination with a pH difference between the buffers, we demonstrated a separation of binary mixtures of pI markers and proteins without applying any external field. The sorting resolution and its efficiency are sufficiently high for sample preparation and could be further improved by optimizing buffers or with an additional transverse electric field. Once fully developed, it can potentially be a pI-based sample fractionation tool for proteomic analysis of complex biomolecule samples.     

Automated 96-well SPE and LC-MS-MS for determination of protease inhibitors in plasma and cartilage tissues

Bioanalytical methods based on automated solid-phase extraction (SPE) and high-performance liquid chromatography with electrospray tandem mass spectrometry (LC-MS-MS) have been developed and utilized for the determination of MMP inhibitors in plasma and cartilage tissues. The SPE methods were automated using a 96-well extraction plate and a 96-channel programmable liquid-handling workstation. The LC-MS-MS methods were developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the positive ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized SPE methods, crude plasma or ground cartilage supernatant samples were loaded onto an SPE plate to remove proteins and other interfering components in the matrixes to render relatively clean extracts for LC-MS-MS analysis. Compared to the simple plasma protein precipitation method, the automated SPE method afforded significant time-saving in sample preparation and improved sensitivity in MS detection. The methods were validated and successfully applied to the analysis of protease inhibitors in plasma and cartilage tissues.     

Fully automated 96-well liquid-liquid extraction for analysis of biological samples by liquid chromatography with tandem mass spectrometry

A fully automated high-throughput liquid-liquid extraction (LLE) methodology has been developed for preparation of biological samples using a 96-well LLE plate and a 96-channel robotic liquid handling workstation. The 96-well LLE plate is made of a 96-well filter plate filled with inert diatomaceous earth particles, allowing continuous and efficient extraction of analytes between the aqueous biological sample and the organic extraction solvent. Two carboxylic acid-based protease inhibitor compounds with high and low levels of plasma protein binding were chosen for the development and application of the automated methodology. The LLE extracts of the plasma samples of the two compounds were analyzed by high-performance liquid chromatography with electrospray (ESI) tandem mass spectrometry (LC-MS/MS). The LC-MS/MS method was developed using a rapid gradient LC separation, followed by sample introduction through an ionspray interface in the negative ion mode and tandem mass spectrometric detection with selected reaction monitoring. In the optimized LLE method, a formate buffer solution was first loaded into a 96-well filter plate packed with inert diatomaceous earth material. Then crude plasma samples and a water-immiscible organic solvent, methyl ethyl ketone, were sequentially added to the LLE plate so that LLE would occur in the interface between the two liquid phases on the surface of individual particles in each well. The organic eluate containing extracted analytes was evaporated and reconstituted for LC-MS/MS analysis. This fully automated LLE methodology avoids several disjointed steps involved in a manual or semiautomated LLE method, leading to significantly reduced sample preparation time, increased sample throughput, and clean sample extracts for improved ESI-MS/MS detection. The automated LLE methodology is universal and can be employed for sample preparation of other biological fluids. The complete bioanalytical method, based on the automated LLE and fast gradient LC-MS/MS, was validated and successfully applied to the quantitative analysis of protease inhibitors in rat plasma.     

Numerical simulation of electroosmotic flow

We have developed a numerical scheme to simulate electroosmotic flows in complicated geometries. We studied the electroosmotic injection characteristics of a cross-channel device for capillary electrophoresis. We found that the desired rectangular shape of the sample plug at the intersection of the cross-channel can be obtained when the injection is carried out at high electric field intensities. The shape of the sample plug can also be controlled by applying an electric potential or a pressure at the side reservoirs. Flow induced from the side channels into the injection channel squeezes the streamlines at the intersection, thus giving a less distorted sample plug. Results of our simulations agree qualitatively with experimental observations.     

Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry

The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (< 5 ppm) in peptide-mapping experiments was obtained by introducing an internal standard via a postseparation channel. The analytical potential of this integrated device for the identification of trace-level proteins from different strains of H. influenzae is demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.     

Focusing of macromolecules using equilibrium between centrifugal and electric force

A focusing separation model for macromolecules has been theoretically investigated. The method involves an ultracentrifugation device, which however, deploys an electric field gradient oriented longitudinally along the radial direction. When a macromolecular sample solution is centrifuged, the molecules which have different density to the surrounding solvent and a non-zero electric charge, experience a combination of centrifugal and electric forces. This forces the molecules to move to the equilibrium positions along the radius of the rotor, which are characterised by the charge over mass ratio of the molecules. Therefore a molecular sample will be separated into its constituents and bands will form, akin to electrophoresis. The bands are, however, focused at their equilibrium position. An example configuration has been examined whereby four proteins with masses between 20 and 100 kDa can be separated within a radial distance of 20 cm, for a rotor spinning at approximately 130,000 rpm and with a varying electric field between 0 and 100 V/cm.     

Monolithic integration of two-dimensional liquid chromatography-capillary electrophoresis and electrospray ionization on a microfluidic device

A microfluidic device capable of two-dimensional reversed-phase liquid chromatography-capillary electrophoresis with integrated electrospray ionization (LC-CE-ESI) for mass spectrometry (MS)-based proteomic applications is described. Traditional instrumentation was used for the LC sample injection and delivery of the LC mobile phase. The glass microfabricated device incorporated a sample-trapping region and an LC channel packed with reversed-phase particles. Rapid electrokinetic injections of the LC effluent into the CE dimension were performed at a cross-channel intersection. The CE separation channel terminated at a corner of the square device, which functioned as an integrated electrospray tip. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. To evaluate the system, LC-MS and LC-CE-MS analyses of protein digests were performed and compared.     

Rectilinear ion trap: concepts, calculations, and analytical performance of a new mass analyzer

A mass analyzer based on a rectilinear geometry ion trap (RIT) has been built, and its performance has been characterized. Design concepts for this type of ion trap are delineated with emphasis on the effects of electrode geometry on the calculated electric field. The Mathieu stability region was mapped experimentally. The instrument can be operated using mass-selective instability scans in both the boundary and resonance ejection versions. Comparisons of performance between different versions of the device having different dimensions allowed selection of an optimized geometry with an appropriate distribution of higher-order electric fields. Comparisons made under the same conditions between the performance of a conventional cylindrical ion trap and a RIT of 4 times greater volume show an improvement of 40 times in the signal-to-noise ratio resulting from the higher ion trapping capacity of the RIT. The demonstrated capabilities of the RIT include tandem mass spectrometry, a mass resolution in excess of 1000, and a mass/charge range of 650 Th, all in a simple structure that is only 3.5 cm(3) in internal volume.     

Interactive control of pulsed field gel electrophoresis via real time monitoring.

The migration of DNA fragment bands through a slab gel can be monitored by UV absorption at 254 nm and imaged by a charge-coupled device (CCD) camera. Background correction and immediate viewing of band positions to interactively change the field program in pulsed-field gel electrophoresis is possible throughout the run via this detection scheme. The use of absorption removes the need for staining or radioisotope labelling, thereby simplifying sample preparation and reducing hazardous waste generation. This leaves the DNA in its native state and further analysis can be performed without destaining. The optimization of buffer concentration, electric field strength, temperature, agarose concentration, as well as pulse duration can considerably reduce total run time. For example, DNA from 2 to 850 kb can be separated in 3 h on a 7-cm gel with interactive control of the pulse time, which is 10 times faster than using a constant field program.     

Effect of a Magnetic Field on the Preparation of Silver Nanowires Using Solid Electrolyte Thin Films

The effect of an external magnetic field on the preparation of silver nanowires was studied. The silver nanowires were synthesized using solid electrolyte RbAg4I5 thin films by applying both a direct current （DC） electric field and a magnetic field. The RbAg4I5 thin films, which were prepared by deposition at room temperature and atmospheric pressure on a NaCI substrate, were used for the transfer of Ag^＋ ions between two Ag electrodes during the preparation process. When only the DC electric field is applied, the silver ions migrate toward the cathode. On the edge of the silver film at the cathode the Ag^＋ ions congregate to form aligned nanowires. If the magnetic field is also applied perpendicular to the DC electric field, the morphology of the nanowires can be controlled by rotating the sample in the magnetic field. Experimental results show that the growth of the silver nanowires is determined by the Ag^＋ ionic flux.     

Toxin screening in phytoplankton: detection and quantitation using MALDI triple quadrupole mass spectrometry

The investigation of a MALDI triple quadrupole instrument for the analysis of spirolide toxins in phytoplankton samples is described in this study. A high-frequency (kHz) laser was employed for MALDI, generating a semicontinuous ion beam, thus taking advantage of the high duty cycle obtained in sensitive triple quadrupole MRM experiments. Initially, several experimental parameters such as type of organic matrix and concentration, solvent composition, and matrix-to-analyte ratio were optimized, and their impact on sensitivity and precision of the obtained ion currents for a reference spirolide, 13-desmethyl-C, was studied. In all quantitative experiments, excellent linearities in the concentration range between 0.01 and 1.75 microg/mL were obtained, with R2 values of 0.99 or higher. The average precision of the quantitative MALDI measurements was 7.4+/-2.4% RSD. No systematic errors were apparent with this method as shown by a direct comparison to an electrospray LC/MS/MS method. Most importantly, the MALDI technique was very fast; each sample spot was analyzed in less than 5 s as compared to several minutes with the electrospray assay. To demonstrate the potential of the MALDI triple quadrupole method, its application to quantitative analysis in several different phytoplankton samples was investigated, including crude extracts and samples from mass-triggered fractionation experiments. 13-Desmethyl spirolide C was successfully quantified in these complex samples at concentration levels from 0.05 to 90.4 microg/mL (prior to dilution to have samples fall within the dynamic range of the method) without extensive sample preparation steps. The versatility of the MALDI triple quadrupole method was also exhibited for the identification of unknown spirolide analogues. Through the use of dedicated linked scan functions such as precursor ion and neutral loss scans, several spirolide compounds were tentatively identified directly from the crude extract, without the usual time-consuming chromatographic preseparation steps. Moreover, high-quality CID spectra were obtained for low-abundant spirolides present in the phytoplankton samples.     

Formation of stable stacking zones in a flow stream for sample immobilization in microfluidic systems

Electrocapture is a multifunctional microfluidic tool that can be used for concentration, sample cleanup, multistep reactions, and separation of biomolecules. Herein, we investigate the mechanisms underlying the electrocapture principle. A microfluidic electrocapture device was found to be capable of generating regions of different electric field, which are maintained in the flow by electric and hydrodynamic forces, with the zones of lower electric field strength upstream of those with higher strength. In addition to detection of the local electric fields by direct measurements, the existence of the zones was observed by the capture of a solution containing Coomassie and myoglobin. The two molecules were captured at different spots in a steady-state manner and were released (separated) at different electric fields. Considering these observations and the experimental values for the electric field strengths, flow velocities, and electrophoretic mobilities of DNA, proteins, and peptides, it is concluded that the macromolecules are captured between the field zones by a stacking mechanism.     

Automated electric valve for electrokinetic separation in a networked microfluidic chip

This paper describes an automated electric valve system designed to reduce dispersion and sample loss into a side channel when an electrokinetically mobilized concentration zone passes a T-junction in a networked microfluidic chip. One way to reduce dispersion is to control current streamlines since charged species are driven along them in the absence of electroosmotic flow. Computer simulations demonstrate that dispersion and sample loss can be reduced by applying a constant additional electric field in the side channel to straighten current streamlines in linear electrokinetic flow (zone electrophoresis). This additional electric field was provided by a pair of platinum microelectrodes integrated into the chip in the vicinity of the T-junction. Both simulations and experiments of this electric valve with constant valve voltages were shown to provide unsatisfactory valve performance during nonlinear electrophoresis (isotachophoresis). On the basis of these results, however, an automated electric valve system was developed with improved valve performance. Experiments conducted with this system showed decreased dispersion and increased reproducibility as protein zones isotachophoretically passed the T-junction. Simulations of the automated electric valve offer further support that the desired shape of current streamlines was maintained at the T-junction during isotachophoresis. Valve performance was evaluated at different valve currents based on statistical variance due to dispersion. With the automated control system, two integrated microelectrodes provide an effective way to manipulate current streamlines, thus acting as an electric valve for charged species in electrokinetic separations.     

Ultratrace LC/MS proteomic analysis using 10-microm-i.d. Porous layer open tubular poly(styrene-divinylbenzene) capillary columns

In this paper, the preparation and performance of long, high-efficiency poly(styrene-divinylbenzene) (PS-DVB), 10-microm-i.d. porous layer open tubular (PLOT) capillary columns are described. PLOT capillaries ( approximately 3% RSD column-to-column retention time), with relatively high permeability, were prepared by in-situ polymerization. Relatively high loading capacities, approximately 100 fmol for angiotensin I and approximately 50 fmol for insulin, were obtained with a 4.2 m x 10-microm-i.d. PLOT column. Low detection levels (attomole to sub-attomole) were achieved when the column was coupled on-line with a linear ion trap MS (LTQ). Analysis of human epidermal growth factor receptor (EGFR), a large transmembrane tyrosine kinase receptor with heterogeneous phosphorylation and glycosylation structures, was obtained at the 25 fmol level. The PLOT column yielded a peak capacity of approximately 400 for the separation of a complex tryptic digest mixture when the sample preparation included a 50-microm-i.d. PS-DVB monolithic precolumn and ESI-MS detection. As an example of the power of the column, 3046 unique peptides covering 566 distinct Methanosarcina acetivorans proteins were identified from a 50 ng in-gel tryptic digest sample combining five cuts in a single LC/MS/MS analysis using the LTQ. The results demonstrate the potential of the PLOT column for high-resolution LC/MS at the ultratrace level.