Capillary array electrophoresis-MALDI mass spectrometry using a vacuum deposition interface.

We previously introduced a vacuum deposition interface for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF MS) on a moving surface (e.g., quartz wheel, Mylar tape, metal target). In our present work, the approach has been extended to demonstrate parallel analysis for multiple on-line infusion MALDI MS and capillary array electrophoresis (CAE)-MALDI MS. In the infusion mode, individual peptide samples were simultaneously deposited on a Mylar tape cartridge using an array of eight capillaries, yielding eight parallel traces. For CAE-MALDI/TOF MS, the same number of separation capillaries were coupled with an array of eight infusion capillaries using a common liquid junction, containing matrix solution. A fast-scanning mirror was employed to traverse the beam of the desorption laser across the Mylar tape to probe one trace at a time. The positions of the eight sample traces formed on the tape were automatically determined, and all samples were analyzed in rapid sequence using a kilohertz repetition rate laser and a high-throughput data acquisition system. The instrumentation was operated with CAE MS for high-throughput analysis without compromising data quality. The principles of parallel separation-vacuum deposition should be generally applicable to MALDI/TOF MS analysis for proteomics and other areas where separation and high throughput are required.     

Characterization of the microdialysis junction interface for capillary electrophoresis/microelectrospray ionization mass spectrometry

A capillary electrophoresis/electrospray ionization mass spectrometry (CE/ESI-MS) interface, based on an electric circuit across a microdialysis membrane surrounding a short capillary segment closely connected to the separation capillary terminus, is demonstrated to be sensitive, efficient, and rugged. A microspray type ionization emitter produces a stable electrospray at the low flow rates provided by CE and thus avoids both the need for a makeup liquid flow provided by liquid junction or sheath flow interfaces and the subsequent dilution and reduction in sensitivity. Reproducibility studies and comparisons with CE/UV and the CE/sheath flow interface with ESI-MS are presented. Additionally, postrun acidification via the microdialysis junction interface is demonstrated and shown to be capable of denaturing the holomyoglobin protein noncovalent complex while maintaining separation efficiency.     

Capillary electrophoresis--matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a vacuum deposition interface

An improved vacuum deposition interface for coupling capillary electrophoresis with MALDI-TOF MS has been developed. Liquid samples consisting of analyte and matrix were deposited on a moving tape in the evacuated source chamber of a TOF mass spectrometer, enabling 24 h of uninterrupted analysis. The vacuum deposition procedure was compared with the dried-droplet method, and it was found that vacuum deposition generated significantly more reproducible signal intensity, eliminating the need for "sweet spot" searching. A concentration detection limit in the low-nanomolar range has been achieved with a low-attomole amount of sample consumed per spectrum. In addition, ion suppression caused by hydrophobicity differences in the analytes was reduced. To minimize ion suppression further, separation prior to MALDI MS analysis was employed. The performance of capillary electrophoresis (CE)-MALDI-TOF MS using the vacuum deposition interface was evaluated with a peptide mixture injected at low-femtomole levels. All peptides were baseline resolved with separation efficiencies in the range of 250000-400000 plates/m (2-3-s band half-width), demonstrating the high separation efficiency of the CE-MALDI MS coupling. A fast (approximately 40 s) CE separation of a mixture of angiotensins was found to reduce significantly ion suppression and enable trace level detection. It was also shown, for the analysis of an enolase digest, that sequence coverage of 65% was obtained using CE separation compared to 52% using step-elution solid-phase extraction and 44% in the control experiment using an unseparated mixture.     

Design and performance of a universal sheathless capillary electrophoresis to mass spectrometry interface using a split-flow technique

A split-flow capillary electrophoresis electrospray ionization mass spectrometry (CE/ESI-MS) interface is introduced, in which the electrical connection to the CE capillary outlet is achieved by diverting part of the CE buffer out of the capillary through an opening near the capillary outlet. The CE buffer exiting the opening contacts a sheath metal tube which acts as the CE outlet/ESI shared electrode. In cases in which the ESI source uses a metal needle, the voltage contact to the CE buffer is achieved by simply inserting the outlet of the CE capillary, which contains an opening, into the existing ESI needle (thereby greatly simplifying the CE to MS interfacing). As a result of the concentration-sensitive nature of ESI, splitting a small percentage of the CE flow has minimal effect on the sensitivity of detection. In addition, because the liquid is flowing through the opening and out of the capillary, there is no dead volume associated with this interface. Moreover, bubble formation due to redox reactions of water at the electrode does not effect CE/ESI-MS performance, because the actual metal/liquid contact occurs outside of the CE capillary. The sensitivity associated with a sheathless CE/MS interface, the ease of fabrication, universality, and lack of any dead volume make this design a superior CE/ESI-MS interface. The performance of this interface is demonstrated by analyses of a peptide standard and a protein digest using a variety of capillary dimensions.     

Capillary electrophoresis coupled with electrochemiluminescence detection using porous etched joint

A new setup to couple capillary electrophoresis (CE) with electrochemiluminescence (ECL) detection is described in which the electrical connection of CE is achieved through a porous section at a distance of 7 mm from the CE capillary outlet. Because the porous capillary wall allowed the CE current to pass through and there was no electric field gradient beyond that section, the influence of CE high-voltage field on the ECL procedure was eliminated. The porous section formed by etching the capillary with hydrofluoric acid after only one side of the circumference of 2-3 mm of polyimide coating of the capillary was removed, while keeping the polyimide coating on the other part to protect the capillary from HF etching makes the capillary joint much more robust since only a part of the circumference of it is etched. A standard three-electrode configuration was used in experiments with Pt wire as a counter electrode, Ag/AgCl as a reference electrode, and a 300-microm diameter Pt disk as a working electrode. Compared with CE-ECL conventional decoupler designs, the present setup with a porous joint has no added dead volume created. Moreover, the dead volume can be increasingly decreased by shortening the distance ( approximately 100 microm) between the working electrode and the end of the separation capillary. The versatility in choice of capillaries and separation buffers within this design is the main advantage over the use of small i.d. capillary and low conductivity buffer in some CE-ECL systems. The performance of this setup is illustrated by the analyses of tripropylamine and proline.     

A sheathless nanoflow electrospray interface for on-line capillary electrophoresis mass spectrometry

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of low-femtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide.     

Capillary electrophoresis time-of-flight mass spectrometry for comparative metabolomics of transgenic versus conventional maize

In this work, capillary electrophoresis time-of-flight mass spectrometry (CE-TOF-MS) is proposed to identify and quantify the main metabolites in three lines of genetically modified (GM) maize and their corresponding nontransgenic parental lines grown under identical conditions. The shotgun-like approach for metabolomics developed in this work includes optimization of metabolite extraction from GM and non-GM maize, separation by CE, online electrospray-TOF-MS analysis, and data evaluation. A large number of extraction procedures and background electrolytes are tested in order to obtain a highly reproducible and informative metabolomic profile. Thus, using this approach, significant differences were systematically observed between the detected amounts of some metabolites in conventional varieties (Aristis, Tietar, and PR33P66 maize) compared with their corresponding transgenic lines (Aristis Bt, Tietar Bt, and PR33P66 Bt maize). Results point to some of these metabolites as possible biomarkers of transgenic Bt maize, although a larger number of samples needs to be analyzed in order to validate this point. It is concluded that metabolomics procedures based on CE-TOF-MS can open new perspectives in the study of transgenic organisms in order to corroborate (or not) their substantial equivalence with their conventional counterparts.     

Hydride generation interface for speciation analysis coupling capillary electrophoresis to inductively coupled plasma mass spectrometry

A novel hydride generation (HG) interface for coupling capillary electrophoresis (CE) with inductively coupled plasma mass spectrometry (ICPMS) is presented in this work. The CE-HG-ICPMS interface was applied to the separation and quantitation of common arsenic species. Lack of a commercially available HG interface for CE-ICPMS led to a three concentric tube design allowing alleviation of back pressure commonly observed in CE-HG-ICPMS. Due to the high sensitivity and element-specific detection of ICPMS, quantitative analysis of As(III), As(V), monomethylarsonic acid, and dimethylarsinic acid was achieved. Optimization of CE separation conditions resulted in the use of 20 mmol L(-1) sodium borate with 2% osmotic flow modifier (pH 9.0) and -20 kV applied potential for baseline resolution of each arsenic species in the shortest time. Hydride generation conditions were optimized through multiple electrophoretic separation analyses with 5% HCl and 3% NaBH(4) (in 0.2% NaOH) determined to be the optimum conditions. After completion of system optimization, detection limits obtained for the arsenic species were less than 40 ng L(-1) with electromigration time precision less than 1% within a total analysis time of 9.0 min. Finally, the interface was used for speciation analysis of arsenic in river and tap water samples.     

Sheathless capillary electrophoresis/electrospray mass spectrometry using a carbon-coated fused-silica capillary

A simple procedure was developed for preparing a carbon-coated fused-silica capillary for use in sheathless capillary electrophoresis/electrospray mass spectrometry (CE/ESI-MS). The tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. The layer from the ink of the marker pen was critical to the preparation of the carbon-coated capillary. The fabrication of a carbon-coated fused-silica capillary tip requires less than 1 min. The stability of this carbon-coated fused-silica capillary is examined, and its utility in on-line sheathless CE/ESI-MS is demonstrated with the separation of berberine, coptisine, and palmatine chlorides. Although the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESI-MS applications. Moreover, it is easy to refurbish the column once the performance of the tip is degraded.     

High capacity capillary electrophoresis-electrospray ionization mass spectrometry: coupling a porous sheathless interface with transient-isotachophoresis

A sheathless interface making use of a porous tip has been used for coupling capillary electrophoresis and electrospray ionization mass spectrometry. First, effective flow rates using the interface have been characterized. It was found that the interface is capable of generating a stable spray with flow rates ranging from below 10 nL/min to >340 nL/min, enabling its use in either the mass or concentration-sensitive region of the electrospray process. Subsequently, by analyzing peptide mixtures of increasing complexity, we have demonstrated that this platform provides exquisite sensitivity enabling the detection of very low amounts of materials with very high resolving power. Transient isotachophoresis (t-ITP) can also be integrated with this setup to increase the mass loading of the system while maintaining peak efficiency and resolution. Concentration limits of detection in the subnanomolar or nanomolar range can be achieved with or without t-ITP, respectively. The application of a vacuum at the inlet of the separation capillary further allowed the peak capacity of the system to be improved while also enhancing its efficiency. As a final step in this study, it was demonstrated that the intrinsic properties of the interface allows the use of coated noncharged surfaces so that very high peak capacities can be achieved.     

Combining liquid chromatography with MALDI mass spectrometry using a heated droplet interface

A novel interfacing technology is described to combine solution-based separation techniques such as liquid chromatography (LC) with matrix-assisted laser desorption ionization (MALDI) mass spectrometry. The interface includes a transfer tube having an inlet and an outlet, the inlet being adapted to accept the LC effluents and the outlet being adapted to form continuously replaced, hanging droplets of the liquid stream, and a MALDI sample plate mounted below the outlet of the transfer tube for collecting the droplets. The liquid stream in the transfer tube is heated to a temperature sufficient to cause partial evaporation of the carrier solvent from the hanging droplets. The droplets are dislodged to the MALDI plate, which is heated to above the boiling point of the carrier solvent to cause further evaporation of the carrier solvent from the collected droplets. It is found that analytes can be fractionated and deposited to a sample spot of 0.8 mm in diameter when a liquid flow rate of up to 50 microL/min and a fractionation interval of 1 min/spot are used. Flow rate of up to 200 microL/min can be used with a deposition sample spot of 2.4 mm in diameter on a commercial MALDI target. This heated droplet interface does not introduce sample loss, and the detection sensitivity of LC/MALDI is similar to that of standard MALDI, i.e., low femtomoles for peptide analysis with a microliter sample deposition. It is compatible with microbore and narrow-bore column separation, thus allowing the injection of a larger amount of sample for separation and analysis, compared to a capillary column LC/MALDI system. The detection dynamic range is shown to be in the order of 10(6) for peptide mixture analysis, which is 4 orders of magnitude greater than standard MALDI. The application of this interface for combining LC with MALDI MS/MS is demonstrated in the proteome analysis of water-soluable protein components of E. coli K12 extracts.     

Characterization of a capillary zone electrophoresis/electrospray-mass spectrometry interface

A dependable and stable CZE/ESI-MS interface has been constructed. To avoid instabilities in both, the capillary electrophoretic separation and the electrospray, the second of the three concentric capillaries in the three-layered sprayer has been replaced by an aluminum-coated fused-silica capillary with an inner diameter only slightly greater than the outer diameter of the separation capillary. By this means, the otherwise often observed destruction of the separation capillary ("electrodrilling") can be avoided completely due to the suppression of electrochemical processes leading to gas bubble formation at the tip of the sprayer. With some examples taken from different biochemical areas and by separation of natural compounds, the capability and the reliability of the modified sprayer as the central part of the interface are demonstrated.     

Capillary electrophoresis, mass spectrometry, and UV-visible absorption studies on electrolyte-induced fractionation of gold nanoclusters

We describe a novel and simple electrolyte-induced fractionation method to separate a polydisperse water-soluble gold nanocluster (Au NC) product. Different particle sizes of Au NC fractions can be easily centrifuged down as a function of the electrolyte concentration or lipophilicity of the solution. The changes in the absorption characteristic of the Au NC fractions under different electrolyte/ethanol conditions demonstrate the change in particle size distribution of the Au NC. Small gold nanoclusters, Au10, Au11, Au12, and Au15, were separated from the Au10-Au50 polydisperse Au NC product under various phosphate/ethanol conditions. The core size separation of Au NC was evaluated by their migration trends in capillary zone electrophoresis, UV-visible absorption, and mass spectra. The electrolyte-induced fractionation not only provides a convenient method to separate small Au NC mixture but also assists in the study of the photophysical properties of smaller Au NCs that are present with the larger Au NCs in a polydisperse Au NC product.     

Simplifying CE-MS operation. 2. Interfacing low-flow separation techniques to mass spectrometry using a porous tip

A robust, reproducible, and single-step interface design between low flow rate separation techniques, such as sheathless capillary electrophoresis (CE) and nanoliquid chromatography (nLC), and mass spectrometry (MS) using electrospray ionization (ESI), is introduced. In this design, the electrical connection to the capillary outlet was achieved through a porous tip at the capillary outlet. The porous section was created by removing 1-1.5 in. of the polyimide coating of the capillary and etching this section by 49% solution of HF until it is porous. The electrical connection to the capillary outlet is achieved simply by inserting the capillary outlet containing the porous tip into the existing ESI needle (metal sheath) and filling the needle with the background electrolyte. Redox reactions of water at the ESI needle and transport of these small ions through the porous tip into the capillary provides the electrical connection for the ESI and for the CE outlet electrode. The etching process reduces the wall thickness of the etched section, including the tip of the capillary, to 5-10 microm, which for a 20-30 microm i.d. capillary results in stable electrospray at approximately 1.5 kV. The design is suitable for interfacing a wide range of capillary sizes with a wide range of flow rates to MS via ESI, but it is especially useful for interfacing narrow (<30 microm i.d.) capillaries and low flow rates (<100 nL/min). The advantages of the porous tip design include the following: (1) its fabrication is reproducible, can be automated, and does not require any mechanical tools. (2) The etching process reduces the tip outer diameter and makes the capillary porous in one step. (3) The interface can be used for both nLC-MS and CE-MS. (4) If blocked or damaged, a small section of the tip can be etched off without any loss of performance. (5) The interface design leaves the capillary inner wall intact and, therefore, does not add any dead volume to the CE-MS or nLC-MS interface. (6) Bubble formation due to redox reactions of water at the high-voltage electrode is outside of the separation capillary and does not affect separation or MS performances. The performance of this interface is demonstrated by the analyses of amino acids, peptide, and protein mixtures.     

A high-efficiency cross-flow micronebulizer interface for capillary electrophoresis and inductively coupled plasma mass spectrometry.

A pneumatic nebulizer interface for capillary electrophoresis (CE) and inductively coupled plasma mass spectrometry (ICPMS) is reported. The interface is constructed using a high-efficiency cross-flow micronebulizer (HECFMN) and has the following features. (1) Makeup solutions can be fed to the interface by nebulizer self-aspiration and liquid gravity pressurization. (2) The liquid dead volume of the interface is approximately 65 nL, much smaller than those (200-2500 nL) reported for other interfaces. (3) The interface can be stably operated at a liquid flow rate down to 5 microL/min with a high analyte transport efficiency up to 95% to the plasma and (4) does not induce noticeable laminar flow in the CE capillary at typical nebulizer gas flow rates of 0.8-1.2 L/min. Because of these features, baseline resolution of 10 lanthanides with a CE-ICPMS system using the HECFMN interface is achieved, and detection limits and peak asymmetry are 0.05-1 microg/L and 0.93-1.23, respectively, improved significantly over those reported previously for a CE-ICPMS system using a high-efficiency nebulizer interface. Peak precision for the 10 lanthanides is in the range of 6.2-12.3% RSD (N = 5). Peak widths are from 9.1 s for 139La to 17.9 s for 175Lu. The effects of nebulizer gas flow rate, makeup solution flow rate, and spray chamber volume on CE-ICPMS signal intensity and separation are also evaluated for the HECFMN interface by the separation of Cr3+ and Cr2O7(2-).     

Determination of bioactive peptides using capillary zone electrophoresis/mass spectrometry.

Mixtures of bioactive peptides have been analyzed by capillary zone electrophoresis/mass spectrometry (CZE/MS) using an on-line coaxial continuous-flow fast atom bombardment interface. High separation efficiencies (up to 410,000 theoretical plates) were obtained from low femtomole levels of peptides. The analysis of basic peptides was accomplished by using aminopropyl-silylated CZE columns to minimize zone broadening due to adsorption effects. CZE/MS/MS data were acquired from femtomole levels of peptides in electrophoretic real time.     

Dating silk by capillary electrophoresis mass spectrometry

A new capillary electrophoresis mass spectrometry (CE-MS) technique is introduced for age estimation of silk textiles based on amino acid racemization rates. With an L to D conversion half-life of ~2500 years for silk (B. mori) aspartic acid, the technique is capable of dating silk textiles ranging in age from several decades to a few-thousand-years-old. Analysis required only ~100 μg or less of silk fiber. Except for a 2 h acid hydrolysis at 110 °C, no other sample preparation is required. The CE-MS analysis takes ~20 min, consumes only nanoliters of the amino acid mixture, and provides both amino acid composition profiles and D/L ratios for ~11 amino acids.     

A low-makeup beveled tip capillary electrophoresis /electrospray ionization mass spectrometry interface for micellar electrokinetic chromatography and nonvolatile buffer capillary electrophoresis

A robust interface has been developed for interfacing micellar electrokinetic chromatography (MEKC) and nonvolatile buffer capillary electrophoresis (CE) to electrospray ionization mass spectrometry (ESI-MS). The interface consists of two parallel capillaries for separation (50 microm i.d. x 155 microm o.d.) and makeup (50 microm i.d. x 155 microm o.d.) housed within a larger capillary (530 microm i.d. x 690 microm o.d.). The capillaries terminate in a single tapered tip having a beveled edge. The use of a tapered beveled edge results in a greater tip orifice diameter (75 microm) than in a previous design from our laboratory (25 microm) that used a flat tip. While maintaining a similar optimum flow rate and consequently similar sample dilution, a 75-microm beveled emitter is more rugged than a 25-microm flat tip. Furthermore, the incorporation of a sheath liquid capillary allows the compositions of the final spray solution to be controlled. The application of this novel CE/ESI-MS interface was demonstrated for MEKC using mixtures of triazines (positive ion mode) and phenols (negative ion mode). The ability to perform CE/ESI-MS using a nonvolatile buffer was demonstrated by the analysis of gangliosides with a buffer consisting of 40 mM borate and 20 mM alpha-cyclodextrin.     

Development of a poly(dimethylsiloxane) interface for on-line capillary column liquid chromatography-capillary electrophoresis coupled to sheathless electrospray ionization time-of-flight mass spectrometry

An interface in elastomeric poly(dimethylsiloxane) (PDMS) for on-line orthogonal coupling of packed capillary liquid chromatography (LC) (i.d. = 0.2 mm) with capillary electrophoresis (CE) in combination with sheathless electrospray ionization (ESI) time-of-flight mass spectrometric (TOFMS) detection is presented. The new interface has a two-level design, which in combination with a continuous CE electrolyte flow through the interface provides integrity of the LC effluent and the CE separation until an injection is desired. The transparent and flexible PDMS material was found to have a number of advantages when combined with fused silica column technology, including ease to follow the process and ease to exchange columns. By combining conventional microscale systems of LC, CE, and ESI-MS, respectively, the time scales of the individual dimensions were harmonized for optimal peak capacity per unit time. The performance of the LC-CE-TOFMS system was evaluated using peptides as model substances. A S/N of about 330 was achieved for leucine-enkephaline from a 0.5 microL LC injection of 25 microg/mL peptide standard.     

Surface plasmon resonance/mass spectrometry interface

A strategy for combining surface plasmon resonance (SPR) biomolecular interaction analysis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported. Both techniques are highly complementary but need separate optimization to improve their individual specificity and sensitivity. Sensor surfaces that are optimal for kinetic analysis are not well suited for MALDI-MS and vice versa. In addition, the transfer of analyte from SPR to MS is crucial and often accompanied by sample loss. To address both of these points, a bifunctional SPR fluid cell was constructed where optimized surfaces can be used for binding studies and MS simultaneously with regard to the special need of each technique. The setup guarantees that the SPR and the loading experiment for MS are performed at identical conditions. A removable pin carries the affinity-surface-bound analyte to the mass spectrometer so that handling is minimized, avoiding analyte elution. Functionalized transfer pins can also be used independently of SPR for microaffinity capture-MS.