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.     

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.     

Enhanced neuropeptide profiling via capillary electrophoresis off-line coupled with MALDI FTMS

An off-line interface incorporating sheathless flow and counter-flow balance is developed to couple capillary electrophoresis (CE) to matrix-assisted laser desorption ionization Fourier transform mass spectrometry (MALDI FTMS) for neuropeptide analysis of complex tissue samples. The new interface provides excellent performance due to the integration of three aspects: (1) A porous polymer joint constructed near the capillary outlet for the electrical circuit completion has simplified the CE interface by eliminating a coaxial sheath liquid and enables independent optimization of separation and deposition. (2) The electroosmotic flow at reversed polarity (negative) mode CE is balanced and reversed by a pressure-initiated capillary siphoning (PICS) phenomenon, which offers improved CE resolution and simultaneously generates a low flow (<100 nL/min) for fraction collection. (3) The predeposited nanoliter volume 2,5-dihydroxybenzoic acid (DHB) spots on a Parafilm-coated MALDI sample plate offers an improved substrate for effective effluent enrichment. Compared with direct MALDI MS analysis, CE separation followed by MALDI MS detection consumes nearly 10-fold less sample (50 nL) while exhibiting 5-10-fold enhancement in S/N ratio that yields the limit of detection down to 1.5 nM, or 75 attomoles. This improvement in sensitivity allows 230 peaks detected in crude extracts from only a few pooled neuronal tissues and increases the number of identified peptides from 19 to 43 (Cancer borealis pericardial organs (n = 4)) in a single analysis. In addition, via the characteristic migration behaviors in CE, some specific structural and chemical information of the neuropeptides such as post-translational modifications and family variations has been visualized, making the off-line CE-MALDI MS a promising strategy for enhanced neuropeptidomic profiling.     

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.     

Identification of proteins in complexes by solid-phase microextraction/multistep elution/capillary electrophoresis/tandem mass spectrometry.

A method to directly identify proteins in complex mixtures by solid-phase microextraction (micro-SPE)/multistep elution/capillary electrophoresis (CE)/tandem mass spectrometry (MS/MS) is described. A sheathless liquid-metal junction interface is used to interface CE and electrospray ionization MS/MS. A subfemtomole detection limit is achieved for protein identification through database searching using MS/MS data. The SPE serves as a semiseparation dimension using an organic-phase step-elution gradient in combination with the second separation dimension for increased resolving power of complex peptide mixtures. This approach improves the concentration detection limit for CE and allows more proteins in complex mixtures to be identified. A 75-protein complex from yeast ribosome is analyzed using this method and 80-90% of the proteins in the complex can be identified by searching the database using the MS/MS data from a complete analysis. This multidimensional CE/MS/MS methodology provides an alternative to multidimensional liquid chromatography/MS/MS for direct identification of small amounts of protein in mixtures.     

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.     

Fully microfabricated and integrated SU-8-based capillary electrophoresis-electrospray ionization microchips for mass spectrometry

We present a fully microfabricated and monolithically integrated capillary electrophoresis (CE)-electrospray ionization (ESI) chip for coupling with high-throughput mass spectrometric (MS) analysis. The chips are fabricated fully of a negative photoresist SU-8 by a standard lithographic process which enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, reproducible analytical performance from chip to chip. As the coaxial sheath flow interface is patterned as an integral part of the SU-8 chip, the fluidic design is dead-volume-free. No significant peak broadening occurs so that very narrow peak widths (down to 2-3 s) are obtained. The sheath flow interface also enables comprehensive optimization of both the CE and the ESI conditions separately so that the same chip design is adaptable to diverse analytical conditions. Plate numbers of the order of 105 m-1 and good resolution are routinely reached for small molecules and peptides within a 2 cm separation length and a typical cycle time of only 30-90 s per sample. In addition, a limit of detection of 100 nM corresponding to a total amount of only 4.5 amol (per injection volume of 45 pL) and excellent quantitative linearity (R2 = 0.9999; 100 nM to 100 microM) were obtained in small-molecule analysis using verapamil as a test compound. The quantitative repeatability was proven good (8.5-21.4% relative standard deviation, peak area) also for the other drug substances and peptides tested.     

Microfabricated devices for capillary electrophoresis-electrospray mass spectrometry.

Two fundamental approaches for the coupling of microfabricated devices to electrospray mass spectrometry (ESI-MS) have been developed and evaluated. The microdevices, designed for electrophoretic separation, were constructed from glass by standard photolithographic/wet chemical etching techniques. Both approaches integrated sample inlet ports, preconcentration sample loops, the separation channel, and a port for ESI coupling. In one design, a modular, reusable microdevice was coupled to an external subatmospheric electrospray interface using a liquid junction and a fused silica transfer capillary. The transfer capillary allowed the use of an independent electrospray interface as well as fiber optic UV detection. In the second design, a miniaturized pneumatic nebulizer was fabricated as an integral part of the chip, resulting in a very simple device. The on-chip pneumatic nebulizer provided control of the flow of the electrosprayed liquid and minimized the dead volume associated with droplet formation at the electrospray exit port. Thus, the microdevice substituted for a capillary electrophoresis instrument and an electrospray interface--traditionally two independent components. This type of microdevice is simple to fabricate and may thus be developed either as a part of a reusable system or as a disposable cartridge. Both devices were tested on CE separations of angiotensin peptides and a cytochrome c tryptic digest. Several electrolyte systems including a transient isotachophoretic preconcentration step were tested for separation and analysis by an ion trap mass spectrometer.     

Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.     

Analysis of inorganic species by capillary electrophoresis-mass spectrometry and ion exchange chromatography-mass spectrometry using an ion spray source

Mixtures of inorganic ions separated by capillary electrophoresis (CE) and ion exchange chromatography (IC) are detected by mass spectrometry (MS) using an ion spray atmospheric pressure ionization source. The selectable degree of ion-adduct declustering and molecular fragmentation in the MS interface region allows the system to be operated as an elemental analyzer or as a molecular detector suitable for oxidation state determinations. Both inorganic anions and cations (including alkalis, alkaline earths, transition metals, and lanthanides) are analyzed by CE-MS. A variety of CE separation buffers are evaluated for the cation analyses (e.g., creatinine, ammonium acetate, and tris[hydroxymethyl]aminomethane). Only one of the buffers (i.e., creatinine) can be used for CE-indirect UV detection. A CE capillary permanently coated with strong anion exchange sites and a pyromellitic acid buffer (suitable for indirect UV detection) is used for the inorganic anion separations. The coated column eliminates the need for buffer modifiers to reverse the flow in the capillary, which then reduces background noise and mass spectral complexity. The separation and detection of 13 inorganic anions are also accomplished by IC using an anion exchange column with a carbonate-bicarbonate mobile phase, on-line suppressed conductivity detection, and mass spectrometric detection.     

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

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

High-throughput microfabricated CE/ESI-MS: automated sampling from a microwell plate

A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is presented. The approach combines a sample-loading port, a separation channel, and a liquid junction, the latter for coupling the device to the MS with a miniaturized subatmospheric electrospray interface. The microdevice was attached to a polycarbonate manifold with external electrode reservoirs equipped for electrokinetic and pressure-fluid control. A computer-activated electropneumatic distributor was used for both sample loading from the microwell plate and washing of channels after each run. Removal of the electrodes and sample reservoirs from the microdevice structure significantly simplified the chip design and eliminated the need both for drilling access holes and for sample/buffer reservoirs. The external manifold also allowed the use of relatively large reservoirs that are necessary for extended time operation of the system. Initial results using this microfabricated system for the automated CE/ESI-MS analysis of peptides and protein digests are presented.     

A colloidal graphite-coated emitter for sheathless capillary electrophoresis/nanoelectrospray ionization mass spectrometry

A colloidal graphite-coated emitter is introduced for sheathless capillary electrophoresis/nanoelectrospray ionization time-of-flight mass spectrometry (CE/ESI-TOFMS). The conductive coating can be produced by brushing the capillary tip to construct a fine layer of 2-propanol-based colloidal graphite. The fabrication involves a single step and requires less than 2 min. Full cure properties develop in approximately 2 h at room temperature and then the tip is ready for use. The coated capillary tip is applied as a sheathless electrospray emitter. The emitter has proven to bear stable electrospray and excellent performance for 50 microm i.d. x 360 microm o.d. and 20 microm i.d. x 360 microm o.d. capillaries within the flow rate of 80-500 nL/min; continuous electrospray can last for over 200 h in positive mode. Baseline separation and structure elucidation of two clinically interesting basic drugs, risperidone and 9-hydroxyrisperidone, are achieved by coupling pressure-assisted CE to ESI-TOFMS using the described sheathless electrospray emitter with a bare fused-silica capillary at pH 6.7. It is found that the signal intensity of m/z in sheathless CE/ESI-TOFMS at pH 6.7 is approximately 50 times higher than that at pH 9.0 for the two analytes, although the electroosmotic flow (EOF) at pH 9.0 provides sufficient flow rate (approximately 150 nL/min) to maintain electrospray.     

Enhancing the coverage of the urinary metabolome by sheathless capillary electrophoresis-mass spectrometry

Sheathless capillary electrophoresis-mass spectrometry (CE-MS), using a porous tip sprayer, is proposed for the first time for highly sensitive metabolic profiling of human urine. A representative metabolite mixture and human urine were used for evaluation of the sheathless CE-MS platform. For test compounds, relative standard deviations (RSDs) for migration times and peak areas were below 2% and 12%, respectively, and an injection volume of only ～8 nL resulted in detection limits between 11 and 120 nM. Approximately 900 molecular features were detected in human urine by sheathless CE-MS whereas about 300 molecular features were found with classical sheath-liquid CE-MS. This difference can probably be attributed to an improved ionization efficiency and increased sensitivity at low flow-rate conditions. The integration of transient-isotachophoresis (t-ITP) as an in-capillary preconcentration procedure in sheathless CE-MS further resulted in subnanomolar limits of detection for compounds of the metabolite mixture, and more than 1300 molecular features were observed in urine. Compared to the classical CE-MS approaches, the integration of t-ITP combined with the use of a sheathless interface provides up to 2 orders of magnitude sensitivity improvement. Hence, sheathless CE-MS can be used for in-depth metabolic profiling of biological samples, and we anticipate that this approach will yield unique information in the field of metabolomics.     

Laser Vaporization/Ionization Interface for Capillary Electrophoresis-Time-of-Flight Mass Spectrometry

Mass spectrometry (MS) is usually coupled on-line with capillary electrophoresis (CE) to analyze biomolecules by using electrospray ionization or continuous-flow fast-atom bombardment. We present a new design for laser vaporization/ionization time-of-flight mass spectrometry. CE, with its low flow rate (<1 μL/min), is highly compatible with MS, even if the total column effluent is introduced directly. A UV laser is used to vaporize and ionize the solution eluting from the column. There is no need to have a makeup solvent. Using this system, we have analyzed a group of amines and peptides. The concentration detection limit of serotonin is in the 10(-)(7) M level. The separation and identification of an amine mixture by CE/MS demonstrates the complementary nature of the information.     

Optimization and evaluation of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry platform for peptide analysis: comparison to liquid chromatography-electrospray ionization mass spectrometry

In this study we have evaluated the suitability of a sheathless capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI-MS) interface with a porous tip as the nanospray emitter for use in peptide analysis. A positively charged capillary coating and 0.1% formic acid as background electrolyte were used for separation upstream from mass spectrometry characterization. The influence of the distance between emitter tip and MS inlet, ESI voltage applied, and of the electroosmotic flow (EOF) on electrospray performance and efficiency of the system was investigated in detail. Under optimized conditions, less than 30 amol of a model peptide (angiotensin I) was required for a detection in the base peak electropherogram and positive identification via tandem MS. Three different cationic capillary coatings were investigated for stability, resolution, and EOF and were found to enable reproducible separations by CE-ESI-MS. After optimizing MS settings, the effectiveness of the CE-ESI-MS method developed was compared with a state-of-the-art nano-liquid chromatography (LC)-ESI-MS method by analyzing Arg-C-digested rat testis linker histones with both systems. With comparable amounts of sample applied, the number of identified peptides increased by more than 60% when using CE-ESI-MS. We found that low molecular mass peptides (below 1400 Da) were preferentially identified by CE-ESI-MS, since this group of peptides poorly interacted with the reversed-phase material in the nano-LC system. Finally, total analysis time in LC-ESI-MS for three runs including equilibration was nearly 4 times longer than that of CE-ESI-MS: 246 versus 66 min.     

Analysis of complex protein mixtures with improved sequence coverage using (CE-MS/MS)n

Identification of proteins, in a complex protein mixture, using one-dimensional high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis of its digest, usually suffers from low sequence coverage. There are several reasons for the low coverage including undersampling, wide concentration dynamic range of the proteins in a complex protein mixture, and wide range of electrospray ionization efficiency of peptides under each mobile-phase composition. To address this low sequence coverage, we introduce a novel technique, (CE-MS/MS)n, which utilizes the most significant advantages of CE-MS/MS, including economy of sample size, fast analysis time, and high separation efficiency, to increase the sequence coverage of a complex protein mixture. Based on these characteristics, (CE-MS/MS)n can be performed in which multiple CE-MS/MS subanalyses (injections followed by analyses) are analyzed and experimental variables are manipulated during each CE-MS/MS subanalysis in order to maximize sequence coverage. (CE-MS/MS)n is a practical technique since each CE-MS/MS subanalysis consumes <10 nL, and each CE-MS/MS subanalysis takes approximately 10 min; therefore, several subanalyses can be performed in approximately 1 h consuming only nanoliters of the sample. Two techniques have been introduced to address the undersampling: (1) (CE-MS/MS)n using dynamic exclusion. In this technique, several CE-MS/MS analyses (injection followed by separation) were performed in one run using the dynamic exclusion capability of the mass spectrometer until all peptide peaks were analyzed by MS/MS. (2) Gas-phase fractionation. In this technique, (CE-MS/MS)n is performed by scanning a narrow mass range (every approximately 100 m/z) during each CE-MS/MS subanalysis without using dynamic exclusion. Under this condition, in each subanalysis, the number of peptides available for MS/MS analysis is significantly reduced, and peptides with the same nominal masses are analyzed, thereby increasing sequence coverage. Additionally, to address the lack of detection of low-level peptides in a mixture containing a wide concentration dynamic range, the concentration of the sample was systematically increased in each subanalysis (while utilizing dynamic exclusion) so that low-intensity peptides would rise above the mass spectrometer threshold and, consequently, undergo MS/MS analysis. Moreover, to alter the ionization efficiency of peptides with low electrospray ionization efficiency, and to change the migration behavior of comigrating peptides under a specific liquid composition, the CE background electrolyte was modified in several subanalyses to further improve sequence coverage. The combination of the above-mentioned techniques was applied to the analysis of the tryptic digests of three well-characterized protein mixtures: a six-protein mixture with average MW of approximately 26,000 (standard I), a six-protein mixture with an average MW approximately 49,000 (standard II), and a more complex protein mixture containing 55 proteins (E. coli ribosomal proteins). In approximately 1 h, when the MS/MS of the peptides were manually checked, all peptides that produced peaks under electrospray ionization in the scanned range of the analysis (500-2000 m/z) and within the practical fragmentation capability of the MS (peptides with MW <3500) were identified for standard I by consuming only 200 fmol of each protein. When searched against a Swissprot database, the average sequence coverage for the standard I, II, and E. coli's ribosomal proteins were 57, 34, and 15%, respectively.     

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.     

Ultra-low flow electrospray ionization-mass spectrometry for improved ionization efficiency in phosphoproteomics

The potential benefits of ultra-low flow electrospray ionization (ESI) for the analysis of phosphopeptides in proteomics was investigated. First, the relative flow dependent ionization efficiency of nonphosphorylated vs multiplyphosphorylated peptides was characterized by infusion of a five synthetic peptide mix with zero to four phophorylation sites at flow rates ranging from 4.5 to 500 nL/min. Most importantly, similar to what was found earlier by Schmidt et al., it has been verified that at flow rates below 20 nL/min the relative peak intensities for the various peptides show a trend toward an equimolar response, which would be highly beneficial in phosphoproteomic analysis. As the technology to achieve liquid chromatography separation at flow rates below 20 nL/min is not readily available, a sheathless capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) strategy based on the use of a neutrally coated separation capillary was used to develop an analytical strategy at flow rates as low as 6.6 nL/min. An in-line preconcentration technique, namely, transient isotachophoresis (t-ITP), to achieve efficient separation while using larger volume injections (37% of capillary thus 250 nL) was incorporated to achieve even greater sample concentration sensitivities. The developed t-ITP-ESI-MS strategy was then used in a direct comparison with nano-LC-MS for the detection of phosphopeptides. The comparison showed significantly improved phosphopeptide sensitivity in equal sample load and equal sample concentration conditions for CE-MS while providing complementary data to LC-MS, demonstrating the potential of ultra-low flow ESI for the analysis of phosphopeptides in liquid based separation techniques.     

A microdevice with integrated liquid junction for facile peptide and protein analysis by capillary electrophoresis/electrospray mass spectrometry

A novel microfabricated device was implemented for facile coupling of capillary electrophoresis with mass spectrometry (CE/MS). The device was constructed from glass wafers using standard photolithographic/wet chemical etching methods. The design integrated (a) sample inlet ports, (b) the separation channel, (c) a liquid junction, and (d) a guiding channel for the insertion of the electrospray capillary, which was enclosed in a miniaturized subatmospheric electrospray chamber of an ion trap MS. The replaceable electrospray capillary was precisely aligned with the exit of the separation channel by a microfabricated guiding channel. No glue was necessary to seal the electrospray capillary. This design allowed simple and fast replacement of either the microdevice or the electrospray capillary. The performance of the device was tested for CE/MS of peptides, proteins, and protein tryptic digests. On-line tandem mass spectrometry was used for the structure identification of the protein digest products. High-efficiency/high-resolution separations could be obtained on a longer channel (11 cm on-chip) microdevice, and fast separations (under 50 s) were achieved with a short (4.5 cm on-chip) separation channel. In the experiments, both electrokinetic and pressure injections were used. The separation efficiency was comparable to that obtained from conventional capillary electrophoresis.