Array of chemically etched fused-silica emitters for improving the sensitivity and quantitation of electrospray ionization mass spectrometry

An array of emitters has been developed for increasing the sensitivity of electrospray ionization mass spectrometry (ESI-MS). The linear array consists of 19 chemically etched fused-silica capillaries arranged with 500 microm (center-to-center) spacing. The multiemitter device has a low dead volume to facilitate coupling to capillary liquid chromatography (LC) separations. The high aspect ratio of the emitters enables operation at flow rates as low as 20 nL/min/emitter, effectively extending the benefits of nanoelectrospray to higher flow rate analyses. To accommodate the larger ion current produced by the emitter array, a multicapillary inlet to the mass spectrometer was also constructed. The inlet, which matched the dimensions of the emitter array, preserved ion transmission efficiency. Standard reserpine solutions of varying concentration were electrosprayed at 1 microL/min using the multiemitter/multi-inlet combination, and the results were compared to those from a standard, single-emitter configuration. A 9-fold sensitivity enhancement was observed for the multiemitter relative to the single emitter. A bovine serum albumin tryptic digest was also analyzed, and a sensitivity increase ranging from 2.4- to 12.3-fold for the detected tryptic peptides resulted; the varying response was attributed to reduced ion suppression under the nanoESI conditions afforded by the emitter array. An equimolar mixture of leucine enkephalin and maltopentaose was studied to verify that ion suppression is indeed reduced for the multiplexed ESI (multi-ESI) array relative to a single emitter over a range of flow rates.     

Multinozzle emitter arrays for nanoelectrospray mass spectrometry

Mass spectrometry (MS) is the enabling technology for proteomics and metabolomics. However, dramatic improvements in both sensitivity and throughput are still required to achieve routine MS-based single cell proteomics and metabolomics. Here, we report the silicon-based monolithic multinozzle emitter array (MEA) and demonstrate its proof-of-principle applications in high-sensitivity and high-throughput nanoelectrospray mass spectrometry. Our MEA consists of 96 identical 10-nozzle emitters in a circular array on a 3 in. silicon chip. The geometry and configuration of the emitters, the dimension and number of the nozzles, and the micropillar arrays embedded in the main channel can be systematically and precisely controlled during the microfabrication process. Combining electrostatic simulation and experimental testing, we demonstrated that sharpened-end geometry at the stem of the individual multinozzle emitter significantly enhanced the electric fields at its protruding nozzle tips, enabling sequential nanoelectrospray for the high-density emitter array. We showed that electrospray current of the multinozzle emitter at a given total flow rate was approximately proportional to the square root of the number of its spraying-nozzles, suggesting the capability of high MS sensitivity for multinozzle emitters. Using a conventional Z-spray mass spectrometer, we demonstrated reproducible MS detection of peptides and proteins for serial MEA emitters, achieving sensitivity and stability comparable to the commercial capillary emitters. Our robust silicon-based MEA chip opens up the possibility of a fully integrated microfluidic system for ultrahigh-sensitivity and ultrahigh-throughput proteomics and metabolomics.     

High-efficiency nanoscale liquid chromatography coupled on-line with mass spectrometry using nanoelectrospray ionization for proteomics

We describe high-efficiency (peak capacities of approximately 10(3)) nanoscale (using column inner diameters down to 15 microm) liquid chromatography (nanoLC)/low flow rate electrospray (nanoESI) mass spectrometry (MS) for the sensitive analysis of complex global cellular protein enzymatic digests (i.e., proteomics). Using a liquid slurry packing method with carefully selected packing solvents, 87-cm-length capillaries having inner diameters of 14.9-74.5 microm were successfully packed with 3-microm C18-bonded porous (300-A pores) silica particles at a pressure of 18,000 psi. With a mobile-phase delivery pressure of 10,000 psi, these packed capillaries provided mobile-phase flow rates as low as approximately 20 nL/min at LC linear velocities of approximately 0.2 cm/s, which is near optimal for separation efficiency. To maintain chromatographic efficiency, unions with internal channel diameters as small as 10 microm were specially produced for connecting packed capillaries to replaceable nanoESI emitters having orifice diameters of 2-10 microm (depending on the packed capillary dimensions). Coupled on-line with a hybrid-quadrupole time-of-flight MS through the nanoESI interface, the nanoLC separations provided peak capacities of approximately 10(3) for proteome proteolytic polypeptide mixtures when a positive feedback switching valve was used for quantitatively introducing samples. Over a relatively large range of sample loadings (e.g., 5-100 ng, and 50-500 ng of cellular proteolytic peptides for 14.9- and 29.7-microm-i.d. packed capillaries, respectively), the nanoLC/nanoESI MS response for low-abundance components of the complex mixtures was found to increase linearly with sample loading. The nanoLC/nanoESI-MS sensitivity also increased linearly with decreasing flow rate (or approximately inversely proportional to the square of the capillary inner diameter) in the flow range of 20-400 nL/min. Thus, except at the lower loadings, decreasing the separation capillary inner diameter has an effect equivalent to increasing sample loading, which is important for sample-limited proteomic applications. No significant effects on recovery of eluting polypeptides were observed using porous C18 particles with surface pores of 300-A versus nonporous particles. Tandem MS analyses were also demonstrated using the high-efficiency nanoLC separations. Chromatographic elution time, MS response intensity, and mass measurement accuracy was examined between runs with a single column (with a single nanoESI emitter), between different columns (same and different inner diameters with different nanoESI emitters), and for different samples (various concentrations of cellular proteolytic peptides) and demonstrated robust and reproducible sensitive analyses for complex proteomic samples.     

Graphite-coated nanoelectrospray emitter for mass spectrometry

A new, more rapid method for coating nanoelectrospray emitters with graphite is to use a vacuum deposition chamber and a graphite carbon electrode. This method allows for mass production of nanoelectrospray emitters in a short period of time. The emitters are laser-pulled borosilicate glass micropipets and have tapers of around 4 microm i.d. The conductive coating applied to the emitter is only 20-30 nm thick, allowing for optical transparency with the borosilicate emitters. The conductive coating is stable for a number of hours at the high voltages used for nanoelectrospray ionization and is durable in both positive and negative ion modes-even during electrical discharge. This stability will make it possible to couple these emitters with online separations such as capillary liquid chromatography or capillary electrophoresis.     

Generation of multiple electrosprays using microfabricated emitter arrays for improved mass spectrometric sensitivity

Arrays of microelectrospray emitters were fabricated on polycarbonate substrates using a laser etching technique. Stable multielectrosprays were successfully generated in the liquid flow rate range relevant to mass spectrometric applications. Comparison of electrosprays generated from the microfabricated emitter array and conventional fused-silica capillaries showed similar spray characteristics and reliability. Higher total electrospray ion currents were observed as the number of electrosprays increased at a given total liquid flow rate. Consistent with the theoretical prediction, the total spray current at a constant total liquid flow rate was shown experimentally to be approximately proportional to the square root of the number of electrosprays. It is further projected that when total flow rate is optimized the maximum achievable total current will be proportional to the number of emitters. Evaluation of the multielectrospray device using a triple quadrupole mass spectrometer showed a factor of 2-3 sensitivity enhancement for the spray numbers ranging from two to nine compared to a conventional single electrospray ionization source under the same operating conditions.     

Fabrication of porous polymer monoliths in polymeric microfluidic chips as an electrospray emitter for direct coupling to mass spectrometry

Coupling of polymeric microfluidic devices to mass spectrometry is reported using porous polymer monoliths (PPM) as nanoelectrospray emitters. Lauryl acrylate-co-ethylene dimethacrylate porous polymer monolith was photopatterned for 5 mm at the end of the channel of microfluidic devices fabricated from three different polymeric substrate materials, including the following: poly(dimethylsiloxane) (PDMS), poly(methyl methacrylate) (PMMA), and cyclic olefin copolymer (COC). Spraying directly from the end of the chip removes any dead volume associated with inserted emitters or transfer lines, and the presence of multiple pathways in the PPM prevents the clogging of the channels, which is a common problem in conventional nanospray emitters. Spraying from a microfluidic channel having a PPM emitter produced a substantial increase in TIC stability and increased sensitivity by as much as 70x compared to spraying from an open end chip with no PPM. The performance of PPM emitter in COC, PMMA, and PDMS chips was compared in terms of stability and reproducibility of the electrospray. COC chips showed the highest reproducibility in terms of chip-to-chip performance, which can be attributed to the ease and reproducibility of the PPM formation due to the favorable optical and chemical properties of COC. We have further tested the performance of the COC chips by constant infusion of poly(propylene glycol) solution at organic content ranging from 10 to 90% methanol and at flow rates ranging from 50 to 1000 nL/min, showing optimum spraying conditions (RSD < 5%) at 50-70% organic content and at flow rates from 100 to 500 nL/min. The PPM sprayer was also used for protein preconcentration and desalting prior to mass spectrometric detection, and results were comparable with a chip spraying from an electrospray tip.     

Capillary-based multi nanoelectrospray emitters: improvements in ion transmission efficiency and implementation with capillary reversed-phase LC-ESI-MS

We describe the coupling of liquid chromatography (LC) separations with mass spectrometry (MS) using nanoelectrospray ionization (nano-ESI) multiemitters. The array of 19 emitters reduced the flow rate delivered to each emitter, allowing the enhanced sensitivity that is characteristic of nano-ESI to be extended to higher flow rate separations. The signal for tryptic fragments from proteins spiked into a human plasma sample increased 11-fold on average when the multiemitters were employed, due to increased ionization efficiency and improved ion transfer efficiency through a newly designed heated multicapillary MS inlet. Additionally, the LC peak signal-to-noise ratio increased approximately 7-fold when the multiemitter configuration was used. The low dead volume of the emitter arrays preserved peak shape and resolution for robust capillary LC separations using total flow rates of 2 microL/min.     

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.     

Microfabricated monolithic multinozzle emitters for nanoelectrospray mass spectrometry

Mass spectrometry is the enabling technology for proteomics. To fully realize the enormous potential of lab-on-a-chip in proteomics, a major advance in interfacing microfluidics with mass spectrometry is needed. Here, we report the first demonstration of monolithic integration of multinozzle electrospray emitters with a microfluidic channel via a novel silicon microfabrication process. These microfabricated monolithic multinozzle emitters (M3 emitters) can be readily mass-produced from silicon wafers. Each emitter consists of a parallel silica nozzle array protruding out from a hollow silicon sliver with a conduit size of 100 x 10 mum. The dimension and number of freestanding nozzles can be systematically and precisely controlled during the fabrication process. Once integrated with a mass spectrometer, M3 emitters achieved sensitivity and stability in peptide and protein detection comparable to those of commercial silica-based capillary nanoelectrospray tips. These M3 emitters may play a role as a critical component in a fully integrated silicon/silica-based micro total analysis system for proteomics.     

Microfluidic dual emitter electrospray ionization source for accurate mass measurements

A glass microfluidic device with two independent electrospray ionization (ESI) emitters has been designed to sequentially generate ions from different solutions for mass analysis. Rapid modulation between the emitters is accomplished by turning on and off the voltage that simultaneously generates the fluid flow rate and ESI potential. The time required to switch between the two electrospray signals is less than 70 ms. Using the second emitter to introduce a reference compound for internal calibration, accurate mass measurements (less than 3 ppm mass error) were obtained with a time-of-flight mass spectrometer.     

On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry

Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using "wrong-way-round" electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/microL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.     

Integration of microfabricated devices to capillary electrophoresis-electrospray mass spectrometry using a low dead volume connection: application to rapid analyses of proteolytic digests.

This report describes the development of a compact and versatile, micromachined chip device enabling the efficient coupling of capillary electrophoresis to electrospray mass spectrometry (CE-ESMS). On-chip separation provides a convenient means of achieving rapid sample cleanup and resolution of multicomponent samples (typically 2-5 min) prior to mass spectral analysis. A low dead volume connection facilitating the coupling of microfabricated devices to CE-ESMS was evaluated using two different interfaces. The first configuration used disposable nanoelectrospray emitters directly coupled to the chip device via this low dead volume junction, thereby providing rapid separation of complex protein digests. The performance of this interface was compared with that of more traditional configurations using a sheath flow CE-ESMS arrangement where a fused-silica capillary of varying length enabled further temporal resolution of the multicomponent samples. The sensitivity and analytical characteristics of these interfaces were investigated in both negative and positive ion modes using standard peptide mixtures. The separation performance for synthetic peptides using a chip coated with amine reagent ranged from 26,000 to 58,000 theoretical plates for a sheath flow CE-ESMS interface comprising a 15-cm CE column. Replicate injections of a dilution series of peptide standards provided detection limits of 45-400 nM without the use of on-line preconcentration devices. The reproducibility of migration time ranged from 0.9 to 1.5% RSD whereas RSDs of 5-10% were observed on peak areas. The application of these devices for the analysis of protein digests was further evaluated using on-line tandem mass spectrometry.     

Membrane-based emitter for coupling microfluidics with ultrasensitive nanoelectrospray ionization-mass spectrometry

An integrated poly(dimethylsiloxane) (PDMS) membrane-based microfluidic emitter for high-performance nanoelectrospray ionization mass spectrometry has been fabricated and evaluated. The ～100-μm-thick emitter was created by cutting a PDMS membrane that protrudes beyond the bulk substrate. The reduced surface area at the emitter enhances the electric field and reduces wetting of the surface by the electrospray solvent. As such, the emitter enables highly stable electrosprays at flow rates as low as 10 nL/min and is compatible with electrospray solvents containing a large organic component (e.g., 90% methanol). This approach enables facile emitter construction and provides excellent stability, reproducibility, and sensitivity as well as compatibility with multilayer soft lithography.     

Macromolecule analysis based on electrophoretic mobility in air: globular proteins

Globular proteins ranging in molecular mass from 5.7 to 669 kDa were separated and analyzed using an aerosol technique based on the electrophoretic mobility of singly-charged molecular ions in air. The ions were produced by electrospraying and drying 100-nm-diameter droplets of a liquid suspension of the proteins, using ionized air to remove the droplet charge due to the spray process. The electrophoretic mobility was measured using a modified commercial continuous-flow differential mobility analyzer operated near atmospheric pressure. An unmodified commercial condensation particle counter was used for detection. The concentrations analyzed ranged from 0.02 to 200 μg of protein/mL of buffer, with a liquid sample flow rate of approximately 50 nL/min. Sampling time of 3 min was used for each complete distribution measured. The electrophoretic mobilities measured were determined entirely from air flow rates, apparatus geometry, and applied potentials. Results were expressed as electrophoretic mobility equivalent diameters using a Millikan formula.     

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.     

Electrospray ionization with a pointed carbon fiber emitter

A new type of electrospray ionization emitter employing a pointed carbon fiber has been developed for interfacing nanoliquid sampling techniques to mass spectrometry. The pointed carbon fiber protruding from an orifice with a surrounding hydrophobic surface confines a small Taylor cone at the tip, which generates a stable electrospray at the tip point. The small Taylor cone improves the electrospray efficiency thereby enhancing the detection limit. This emitter is rugged and able to generate stable electrospray over a wide range of flow rate, ESI voltage, and surface tension variation. Using a solution of angiotensin I, the carbon fiber emitter in 75-microm-i.d. fused-silica tubing was shown to give ion current comparable to that from a commercial 8 microm orifice nanospray emitter. Use of the emitter for ESI-MS/MS analysis of peptides was examined by infusing a mixture of cytochrome c and myoglobin tryptic digest peptides. Protein identification was demonstrated at the level of less than 1 fmol of the peptide consumed. The use of the carbon fiber emitter for interfacing monolithic capillary HPLC to MS was also demonstrated.     

Porous polymer monolith assisted electrospray

Coupling low-flow analytical separation instrumentation such as capillary electrophoresis, capillary electrochromatography, nano-HPLC, and microfluidic-based devices with electrospray ionization mass spectrometry has yielded powerful analytical tools. However, conventional coupling methodologies such as nanospray suffer from limitations including poor conductive coating robustness, constant clogging, complicated fabrication processes, and incompatibility with large flow rate regimes. This study demonstrates that robust nanospray emitters can be fabricated through the formation and utilization of a porous polymer monolith (PPM) at the end of a fused-silica capillary. Stable electrosprays can be produced from capillaries (75-100-microm i.d.) at a variety of flow rates (50-1000 nL/min) without the need to taper the capillaries by etching or pulling. The PPM is photopatterned to be present only near the capillary exit aperture using conditions that generate pore sizes similar to those seen with nanospray tips. The porous nature of the PPM aids in developing a stable electrospray generating a single clearly visible Taylor cone at relatively high flow rates while at low flow rates (<100 nL/min) a mist, presumably from multiple small Taylor cones, develops. The hydrophobic nature of the PPM should limit problems with band broadening associated with droplet spreading at the capillary exit, while the multiple flow paths inherent in the PPM minimize clogging problems associated with conventional nanospray emitters. Total ion current traces for a constant infusion of standard PPG and cytochrome c solutions are very stable with deviations ranging from only 3 to 8%. The PPM-assisted electrospray produces mass spectra with excellent signal-to-noise ratios from only a few femtomoles of material.     

Rapid prototyping and flow simulation applications in design of agricultural irrigation equipment: Case study for a sample in-line drip emitter

In this study, a 3D CAD solid model of a sample agricultural irrigation emitter was created and the flow behaviour was simulated in its labyrinth channels by using a flow simulation technique. Referenced by the original design, the channel geometry was modified and the emitter was re-fabricated using rapid prototyping/additive manufacturing techniques. The flow behaviour is then re-investigated based on the re-shaped channel geometry of the labyrinth structure. The predetermined optimum pressure in the pipe was validated experimentally for both the original design and modified designs. As a result, the optimum pressure in the pipe and the flow characteristics for original both the design and modified designs of the emitter were validated. This study contributes to further research into the development of agricultural irrigation equipment aided through the utilisation of additive manufacturing and computer aided engineering tools.     

Chemically etched open tubular and monolithic emitters for nanoelectrospray ionization mass spectrometry

We have developed a new procedure for fabricating fused-silica emitters for electrospray ionization-mass spectrometry (ESI-MS) in which the end of a bare fused-silica capillary is immersed into aqueous hydrofluoric acid, and water is pumped through the capillary to prevent etching of the interior. Surface tension causes the etchant to climb the capillary exterior, and the etch rate in the resulting meniscus decreases as a function of distance from the bulk solution. Etching continues until the silica touching the hydrofluoric acid reservoir is completely removed, essentially stopping the etch process. The resulting emitters have no internal taper, making them much less prone to clogging compared to, e.g., pulled emitters. The high aspect ratios and extremely thin walls at the orifice facilitate very low flow rate operation; stable ESI-MS signals were obtained for model analytes from 5-microm-diameter emitters at a flow rate of 5 nL/min with a high degree of interemitter reproducibility. In extensive evaluation, the etched emitters were found to enable approximately four times as many LC-MS analyses of proteomic samples before failing compared with conventional pulled emitters. The fabrication procedure was also employed to taper the ends of polymer monolith-containing silica capillaries for use as ESI emitters. In contrast to previous work, the monolithic material protrudes beyond the fused-silica capillaries, improving the monolith-assisted electrospray process.     

Very high pressure gradient LC/MS/MS

A very high pressure liquid chromatography (VHPLC) system was constructed by modifying a commercially available pump in order to achieve pressures in excess of 1,200 bar (17,500 psi). A computer-controlled low-pressure mixer was used to generate solvent gradients. Protein digests were rapidly analyzed by reversed-phase VHPLC with linear solvent gradients coupled to either a tandem mass spectrometer using electrospray ionization or a UV/visible detector. The separations were performed at pressures ranging from 790 (11,500 psi) to 930 bar (13,500 psi) in 22-cm-long capillary columns packed with C18-modified 1.5-microm nonporous silica particles. A digest of bovine serum albumin (BSA) was analyzed by the VHPLC system connected to a mass spectrometer in MS mode. An analysis of 12.5 fmol of sample gave signal-to-noise ratios of tryptic peaks greater than 10:1 in the base peak plot mass chromatogram. This system was also used to analyze a proteolytic digest of a rat liver protein excised from a 2-D gel separation of a liver tissue lysate. For this analysis, the mass spectrometer was set up to perform data-dependent scanning (automated switching from MS mode to MS/MS mode when a peak was detected) for peptide sequencing and protein identification by database searching. The results of this analysis are compared to an analysis performed on the same sample using the nanoelectrospray-MS/MS technique. Though both techniques were able to identify the unknown protein, the VHPLC method gave twice as many sequenced peptides as nanoelectrospray and improved the signal-to-noise ratio of the spectra by at least a factor of 10. Direct comparisons with nanoelectrospray for MS and MS/MS data acquisition from a BSA digest were made. These comparisons show enhancements of greater than 20-fold for VHPLC over nanoelectrospray. In addition, the VHPLC/MS/MS data acquisition was accomplished in an automated manner.