A microchip emitter for solid phase extraction - gradient elution - mass spectrometry (original) (raw)
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Microchip emitter for solid-phase extraction-gradient elution-mass spectrometry
Analytical chemistry, 2013
A microchip electrospray emitter with a magnetic bead trap has been designed for solid-phase extraction-gradient elution-mass spectrometry (SPE-GEMS). The goal of this method is the detection of analytes at low concentrations and it is here demonstrated using reverse phase coated magnetic beads (Mbs) for the preconcentration and detection of the peptides. The sample is passed through the chip, and the peptides are retained and enriched in the trap. After washing, the peptides are released sequentially by stepwise gradient elution and electrosprayed for mass spectrometry analysis. This approach allows effective sample desalting, enrichment, sequential elution, and MS detection without the introduction of an additional separation step after SPE. Efficient preconcentration of model peptides by SPE and sequential release and analysis of peptides by GEMS were demonstrated for diluted sample solutions within the range of 1 μM to 10 nM. Fortified human blood serum, protein digest and fractions collected after protein digest OFFGEL separation were analyzed by SPE-GEMS allowing the detection of low abundance peptides usually not observed by direct mass spectrometry analysis. A mathematical model for gradient elution is proposed.
Journal of Chromatography A, 2004
Field-enhanced sample injection (FESI) was used to improve the concentration sensitivity of a capillary electrophoresis (CE)-mass spectrometry (MS) system with sheath flow configuration. Using some bioactive peptides, more than 3000-fold improvement in signal was obtained, permitting analysis in the low nM (fmol/l) levels. The system was further evaluated for analysis of complex peptide mixtures by using low concentration tryptic digests of standard proteins. Rapid identification of the original protein was obtained by database searching using the observed molecular masses of the peptides, and by comparison of actual MS-MS spectra of selected peptides with the predicted fragmentation patterns.
Analytical Chemistry, 2006
A versatile experimental approach is described to achieve very high sensitivity analysis of peptides by capillary electrophoresis-mass spectrometry with sheath flow configuration based on optimization of field-amplified sample injection. Compared to traditional hydrodynamic injection methods, signal enhancement in terms of detection sensitivity of the bioanalytes by more than 3000-fold can be achieved. The effects of injection conditions, composition of the acid and organic solvent in the sample solution, length of the water plug, sample injection time, and voltage on the efficiency of the sample stacking have been systematically investigated, with peptides in the lownanomolar (10 -9 M) range readily detected under the optimized conditions. Linearity of the established stacking method was found to be excellent over 2 orders of magnitude of concentration. The method was further evaluated for the analysis of low concentration bioactive peptide mixtures and tryptic digests of proteins. A distinguishing feature of the described approach is that it can be employed directly for the analysis of lowabundance protein fragments generated by enzymatic digestion and a reversed-phase-based sample-desalting procedure. Thus, rapid identification of protein fragments as low-abundance analytes can be achieved with this new approach by comparison of the actual tandem mass spectra of selected peptides with the predicted fragmentation patterns using online database searching algorithms.
Journal of Chromatography A, 2007
The use of solid-phase extraction coupled on-line to capillary electrophoresis using electrospray mass spectrometry detection (SPE-CE-ESI-MS) is described for the analysis of peptides in dilute solutions. A SPE microcartridge or analyte concentrator containing C 18 derivatized silica particles as the extraction sorbent was easily constructed near the inlet of the separation capillary using commercially available materials. The reversed-phase sorbent selectively retained the target peptides, enabling large volumes of the sample to be introduced (>100 L). The captured analytes were eluted in a small volume of an appropriate solution (20-50 nL). This resulted in sample clean-up and concentration enhancement, with minimum sample handling. As the SPE-CE conditions were compatible with on-line ESI-MS detection, the potential for identifying and characterizing the preconcentrated analytes by SPE-CE-ESI-MS using a sheath-flow CE-ESI-MS interface is also shown. Using separation electrolytes containing N-[carbamoylmethyl]-2-aminoethanesulfonic acid (ACES) at pH 7.4, an elution plug of 80:20 (v/v) (25 mM of formic acid in MeCN):H 2 O and a sheath liquid of 20 mM of acetic acid in 50:50 (v/v) methanol:H 2 O the concentration limits of detection for the analyzed peptides in the positive ion mode were lowered to nanogram per milliliter levels. The systematic optimization of the operational parameters involved in the development of the SPE-CE method is described in detail, in order to promote robust and quantitative SPE-CE-ESI-MS analysis and facilitate the widespread use of the technique.
Analytical Chemistry, 2000
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 (halfheight 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 demon-strated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.
Time-of-flight mass spectrometry (TOFMS) is coupled online with capillary electrophoresis (CE) to analyze mixtures of biomolecules using an electrospray ionization (ESI) interface. The eluent is electrosprayed directly from the CE capillary using a thin gold wire to maintain its potential. The ions are extracted at right angles to the initial direction of the ion beam, and a complete mass spectrum is recorded every 100 ~s . This CE/ESI-TOFMS apparatus achieves a separation efficiency of 50 000 theoretical plates with a concentration detection limit of (1-2) X molar, which corresponds to a mass detection limit of approximately 40-80 fmol per component.
Analytical Chemistry, 2010
In this paper, we are evaluating the strategy of sorting peptides / proteins based on the charge to mass without resorting to ampholytes and / or isoelectric focusing, using a single-and two-step free-flow zone electrophoresis. We developed a simple fabrication method to create a salt bridge for free-flow zone electrophoresis in PDMS chips by surface printing a hydrophobic layer on a glass substrate. Since the surface-printed hydrophobic layer prevents plasma bonding between the PDMS chip and the substrate, an electrical junction gap can be created for free-flow zone electrophoresis. With this device, we demonstrated a separation of positive and negative peptides and proteins at a given pH in standard buffer systems, and validated the sorting result with LC/MS. Furthermore, we coupled two sorting steps via off-chip titration, and isolated peptides within specific pI ranges from sample mixtures, where the pI range was simply set by the pH values of the buffer solutions. This free-flow zone electrophoresis sorting device, with its simplicity of fabrication, and a sorting resolution of 0.5 pH unit, can potentially be a high-throughput sample fractionation tool for targeted proteomics using LC/MS.
Analytical Chemistry, 2003
Although several designs have been advanced for coupling sample enrichment devices to a sheathless electrospray ionization-mass spectrometry (MS) interface on a capillary electrophoresis (CE) column, most of these approaches suffer from difficulties in fabrication, and the CE separation efficiency is degraded as a result of the presence of coupling sleeves. We have developed a design that offers significant improvements in terms of ease of fabrication, durability, and maintenance of the integrity of the CEseparated analyte zones. Capillaries with different inside and outside diameters were evaluated to optimize the performance of the CE-MS system, resulting in a mass limit of detection of 500 amol for tandem MS analysis of a standard peptide using a 20-µm-i.d. capillary. The improved design incorporates an efficient method to preconcentrate a sample directly within the CE capillary followed by its electrophoretic separation and detection using a true zero dead-volume sheathless CE-MS interface. Testing of this novel CE-MS system showed its ability to characterize proteomic samples such as protein digests, in-gel-digested proteins, and hydrophobic peptides as well as to quantitate ICAT-labeled peptides.
The separation of peptide mixtures from proteolytic cleavage is often necessary prior to mass spectrometry (MS) to enhance sensitivity and peptide mapping coverage. When buffers, salts, and other higher abundance peptides/contaminants are present, competition for charge during the electrospray ionization and matrix-assisted laser desorption/ionization (MALDI) processes can lead to ion suppression for the targeted analyte(s). In this note, a simple reversed-phase microcolumn sample separation and deposition device (Sep-Dep) is described. The use of this device improves or renders possible the analysis of complex or contaminated peptide mixtures by MALDIMS. The method is simple and inexpensive and utilizes single-use low-cost Geloader-type columns packed with reversed-phase material. The device described utilizes an open column, allowing for a gradient or narrow-step gradient to be applied by any solvent delivery system or manually with a pipet. A key feature of the device is a deposition chamber that can be custom-built to hold any MALDI target. The Sep-Dep device is attached directly to an in-house vacuum line and draws solvent from the open-ended LC column. The elution of separated peptides is performed directly onto a target that has been treated with a hydrophobic barrier. This barrier effectively isolates fractions and improves the quality and morphology of the matrix crystals. The method produces efficient separations of proteolytic peptides, significantly reducing signal suppression effects in MALDI.