Electrospray Ionization with a Pointed Carbon Fiber Emitter - American

173 Ashley Avenue, Charleston, South Carolina 29425. A new type of electrospray ionization emitter employing a pointed carbon fiber has been developed...
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Anal. Chem. 2004, 76, 3599-3606

Electrospray Ionization with a Pointed Carbon Fiber Emitter Jian Liu, Kyung Won Ro, Mark Busman, and Daniel R. Knapp*

Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, Charleston, South Carolina 29425

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-µm-i.d. fused-silica tubing was shown to give ion current comparable to that from a commercial 8 µm 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. The development of highly sensitive methods for analysis of peptides and proteins by mass spectrometry and tandem mass spectrometry has had a vital role in the expansion of the field of proteomics. With the development of proteomics, the need for detecting low-abundance proteins in a complex matrix becomes more significant, and interfacing high-resolution separation to mass spectrometry is key to meeting this requirement. Electrospray ionization is one of the major methods for generating charged ions of proteins and peptides for mass spectrometric analysis. The application of ESI-MS to biological studies has been aided by the development of low flow rate emitters,1-3 which have advantages in terms of absolute amount of sample required. Sheathless ESI is often adopted when interfacing low flow rate infusion or low flow rate separation techniques such as capillary electrophoresis (CE), capillary electrochromatography, and nano* To whom correspondence should be addressed. E-mail: [email protected]. Fax: 843-792-2475. (1) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180. (2) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (3) Gale, D. C.; Smith, R. D. Rapid Commum. Mass Spectrom. 1993, 7, 10171021. 10.1021/ac030419i CCC: $27.50 Published on Web 05/29/2004

© 2004 American Chemical Society

liquid chromatography to MS.4 Those interfaces require that the emitters have high electrospray efficiency and low sample consumption. Low flow rate ESI emitters are traditionally fabricated with use of a capillary of small exit aperture. A convenient method for the fabrication is by “pulling” glass or fused-silica tubing. Recently, novel techniques have been utilized to manufacture microfluidic devices with ESI emitters possessing the attractive characteristics inherent to traditional low-flow emitters. Electrospray can be generated from an open channel with a sharp exit on a PMMA chip.5 It appeared in this work that the width of the channel exit determined the dimensions of the Taylor cone rather than the crude saw cut sharp tip since no difference was observed with different tip angles. Recently, Hsu et al.6 interfaced thin-layer chromatography to ESI-MS. When small TLC strips with a sharp end were used for electrospray ionization, they observed the Taylor cone formed around the sharpened tip of the TLC strip rather than the whole alumina triangle. Kameoka et al.7 sandwiched a triangularly shaped hydrophilic parylene C film between two pieces of cycloolefin substrates. The liquid from the channel outlet wetted the two-dimensional triangle tip. Due to the contact angle of the buffer solution on the cycloolefin, the liquid was completely confined on the triangular tip and generated a stable electrospray. The outlet walls of hydrophobic polymer avoided the wetting of the spray edge and generated an efficient nanospray at the sharply trimmed tip.8 The high voltage for electrospray ionization can be applied internally (i.e., via the sample liquid) or externally. Generally, conductive material coated-nanoemitters employ high voltage applied externally. As an exception, Herring and Qin9 demonstrated an ESI interface for CE/MS where the ESI voltage was applied through a palladium wire inserted into the emitter tip. This interface generated some turbulence in the capillary, thus degrading resolution to a small extent. As an example of internal high-voltage application, Chiou et al.10 integrated a sample reservoir, a microchannel, a built-in electrode, and a silica capillary (4) Nilsson, S.; Svedberg, M.; Pettersson, J.; Bjo¨refors, F.; Markides, K.; Nyholm, L. Anal. Chem. 2001, 73, 4607-4616. (5) Yuan, C. H.; Shiea, J. Anal. Chem. 2001, 73, 1080-1083. (6) Hsu, F. L.; Chen, C. H.; Yuan, C. H.; Shiea, J. Anal. Chem. 2003, 75, 24932498. (7) Kameoka, J.; Orth, R.; Ilic, B.; Czaplewski, D.; Wachs, T.; Craighead, H. G. Anal. Chem. 2002, 74, 5897-5901. (8) Gobry, V.; Oostrum, J. V.; Martinelli, M.; Rohner, T. C.; Reymond, F.; Rossier, J. S.; Girault, H. Proteomics 2002, 2, 405-412. (9) Herring, C. J.; Qin, J. Rapid Commun. Mass Spectrom. 1999, 13, 1-7. (10) Chiou, C. H.; Lee, G. B.; Hsu, H. T.; Chen, P. W.; Liao, P. C. Sens. Actuators, B 2002, 86, 280-286.

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nozzle in a small PDMS chip. The high voltage for generating electrospray was applied via the built-in electrode. In Cao and Moini’s design,11 electrical contact was achieved and maintained by placing the electrode inside the CE capillary through a small hole in the capillary wall near the terminus. The electrode acted both as the low-voltage electrode of the CE electric circuit and as a connection for the ESI voltage. With no metal at the tip, the capillary terminus could be placed closer to the inlet of the mass spectrometer without arcing. A special case for internal highvoltage application is open-channel ESI, where the high-voltage input point is away from the emitter tip.5 Emitters with small-diameter apertures (50 µm) are easy to maintain. However, they are not normally able to produce the efficient ESI behavior inherent to the smaller aperture emitters and, consequently do not achieve the detection limits of smaller aperture emitters.12 An integral part of many low flow rate emitters is a conductive coating applied to the outer surface of the spraying device. Such conductive coatings are often necessary for the application of ESI voltages in devices constructed from insulating materials such as glass and fused silica. Coating methods have included vapor deposition of a metal layer, painting of conductive materials, and coating with finely divided conductive particles. Generally, the sputter-coated noble metal layer on the emitter tip of a tapered fused-silica capillary deteriorates quickly. This hampers achieving long-term electrospray stability and makes routine applications of sheathless ESI-MS difficult. The deterioration of a sputter-coated gold layer can be attributed to the electrical discharge, electrochemical reactions, and poor mechanical adhesion to the fused-silica surface. The sputter-coated gold layer on the emitter may peel off because of mechanical stress caused by the gas evolution accompanying water oxidation or reduction. Emitters with gold coatings on top of adhesion layers of chromium and nickel alloy withstand this mechanical stress and have excellent durability when operating as cathodes. When operating as anodes, the adhesion layer is electrochemically dissolved through the gold film, which results in the flaking away of the gold film.4 Sputter coating followed by electroplating of gold can significantly extend the lifetime of a tapered emitter.13 The coating of gold films on a titanium adhesion layer on an ESI emitter has been demonstrated to be durable. Despite the fact that the titanium layer did not undergo anodic dissolution, it was found that the emitter still failed in electrochemical tests at potentials where water was oxidized. The titanium adhesion layer could not prevent the gold film from detaching.14 The gold layer can be overcoated with an inert, electrochemically stable dielectric layer, locking the gold into place. The resulting coating has excellent resistance to ESI solvent exposure and exceptional electrochemical (11) Cao, P.; Moini, M. J. Am. Soc. Mass Spectrom. 1997, 8, 561-564. (12) Janini, G. M.; Conrads, T. P.; Wilkens, K. L.; Issaq, H. J.; Veenstra, T. D. Anal. Chem. 2003, 75, 1615-1619. (13) Barroso, M. B.; de Jong; Ad. P. J. Am. Soc. Mass Spectrom. 1999, 10, 12711278. (14) Nilsson, S.; Klett, O.; Svedberg, M.; Amirkhani, A.; Nyholm, L. Rapid Commun. Mass Spectrom. 2003, 17, 1535-1540.

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stability.15 Gold layer deterioration, to a certain extent, seems related to the properties of the sputter-coated gold layer. To improve the properties of electrospray emitters, alternative conductive coatings have been evaluated. Colloidal graphite has been used as a convenient coating material for electrospray emitter fabrication. Colloidal graphite-coated emitters have shown a long lifetime and allow stable ESI operation.16 A carbon-coated capillary has been shown to be useful for microspray and sheathless CE/ ESI-MS applications.17 A tapered capillary tip was smeared with a marker pen before coating with carbon using a soft pencil. While the carbon-coated fused-silica capillary tip is not as rugged as a gold-coated capillary, it is durable enough for sheathless CE/ESIMS applications. In further refinement of the carbon coating fabrication process, a carbon-coated bevel-tapered capillary tip was developed.18 The optimal flow rate and the sensitivity of a beveled 75-µm-i.d. tapered tip was found to be similar to a 25-µm-i.d. flat tip. However, the beveled tapered capillary tip was shown to be more durable than the 25-µm-i.d. tapered capillary because of the larger size. It was shown that the shape of the tip plays a key role in electrospray. The beveled tip showed good spray behavior over a wide flow rate range. Wetterhall et al.19 compared nanospray emitters based on chronoamperometric experiments in the region of water oxidation to qualitatively estimate tip stability in positive ESI. The chronoamperometric results indicated that the graphitecoated nanospray emitters show better electrochemical stabilities than the corresponding noble metal-coated emitters. In this work, we describe a new micro ESI emitter based upon a carbon fiber that can eliminate the clogging problem and restrict the contact between sample liquid and gold layer. This emitter design can enhance durability and reliability and extend the emitter’s lifetime and applicability. It achieved high electrospray ionization efficiency comparable to conventional nanoemitters and can be used to interface microcolumn liquid chromatography to mass spectrometry. METHODS Materials and Chemicals. Trypsin (proteomics grade), myoglobin (horse heart), cytochrome c (horse heart), dithiothreitol, and albumin (bovine, BSA) were purchased from Sigma (St. Louis, MO). Trifluoroacetic acid, acetonitrile, methanol, basic alumina, ammonium bicarbonate, and trichloroacetic acid were obtained from Fisher Scientific (Pittsburgh, PA) and used without further purification. Double-distilled water was used. Iodoacetamide, lauryl methacrylate (LMA), ethylene glycol dimethacrylate (EDMA), 1-propanol, 1,4-butanediol, and 2,2-dimethoxy-2-phenylacetophenone (DAP) were purchased from Aldrich (Milwaukee, WI). LMA and EDMA were purified by passing through basic alumina to remove inhibitors. Fused-silica capillaries (75-µm i.d., 363-µm o.d.) with polyimide and (100-µm i.d., 363-µm o.d.) with UV-transparent fluorinated hydrocarbon polymer coatings were obtained from Polymicro Technologies (Phoenix, AZ). Carbon fibers (30-µm diameter, 2-m length) were obtained from World Precision (15) Valaskovic, G. A.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1996, 7, 1270-1272. (16) Zhu, X.; Thiam, S.; Valle, B. C.; Warner I. M. Anal. Chem. 2002, 74, 54055409. (17) Chang, Y. Z.; Her, G. R. Anal. Chem. 2000, 72, 626-630. (18) Chang, Y. Z.; Chen, Y. R.; Her, G. R. Anal. Chem. 2001, 73, 5083-5087. (19) Wetterhall, M.; Klett, O.; Markides, K. E.; Nyholm L.; Bergquist, J. Analyst 2003, 128, 728-733.

Instruments (Sarasota, FL). Carbon ink adhesive was from Conductive Compounds, Inc. (Londonderry, NH). Perfluoroalky ether was from Dupont (Wilmington, DE). Multilayer platinumcoated Pico Tip nanospray emitters (8-µm i.d.) were obtained from New Objective (Woburn, MA). Protein Digestion. Myoglobin (0.5 mg) and cytochrome c (0.35 mg) were dissolved in 1.0 mL of water. The protein solution was denatured in a boiling water bath for 30 min. Trypsin (20 µg) was dissolved in 400 µL of NH4HCO3 solution (100 mmol). Trypsin solution (200 µL) was added to the denatured protein solution. Digestion was performed at 37 °C for 24 h. The digestion was quenched by addition of 10 µL of trifluoroacetic acid (TFA). The digest was desalted using HPLC. The eluted peptides were stepwise diluted to 10 fmol/µL with makeup solution of MeOH/ H2O/HOAc (48:48:4 by volume). BSA (1.0 mg) was dissolved into 1.0 mL of NH4HCO3 solution (50 mmol). The BSA was reduced at 56 °C for 1 h by the addition of 460 µg of dithiothreitol (in 0.5 mL of NH4HCO3 solution). The reduced protein was alkylated with 3.1 mg of iodoacetamide in dark conditions at ambient temperature for 45 min. The alkylated BSA protein was precipitated in 10% TCA water solution at 0 °C for 30 min. The precipitant was centrifuged, and the supernatant was removed. The pellet was washed twice with double-distilled water. The washed pellet was dissolved in 1.0 mL of NH4HCO3 solution (50 mmol). Trypsin (10 µg in 50 mmol of NH4HCO3 solution) was added, and the protein was digested at 37 °C overnight. The digestion was quenched by addition of 10 µL of TFA. The digest was desalted on a C18 solid-phase extraction cartridge. Fabrication of Carbon Fiber (CF) Emitter. CF emitters were constructed from lengths of fused-silica capillary tubing. At the emitter terminus of the capillary, a 5-mm length of polyamide coating was removed by burning in a flame. A carbon fiber was inserted 5 mm into the capillary. The position of the fiber was fixed with a small “dot” of carbon ink adhesive. The carbon fiber was allowed to protrude ∼0.2 mm from the tubing terminus. The protruding carbon fiber was etched to a pointed shape by application of a discharge from a model BD-20 Tesla coil (ElectroTechnic Products, Chicago, IL). Upon etching, the pointed carbon fiber is observed to protrude ∼20-30 µm from the tubing terminus. The assembly was sputter-coated with a layer of gold utilizing a model E5150 (Polaron Equipment Ltd., Doylestown, PA) SEM coating unit for 10 min at 2-kV voltage dc glow discharge. The gold emitter terminus was dipped into Krytox 1625 perfluoralkyl ether oil (Dupont, Wilmington, DE) with a flow of nitrogen through the tubing to prevent wetting the inside and then allowed to drain excess Krytox for 1 min on a piece of absorbent tissue, leaving a thin hydrophobic film on the outside of the tubing. Mass Spectrometry. Electrospray ionization mass spectrometry was performed on a ThermoFinnigan (San Jose, CA) LCQ Classic ion trap mass spectrometer fitted with a custom X, Y, Z adjustable source platform. The source platform was used for evaluation of the metal, nanospray, and CF emitters. The source platform facilitates the precise adjustment of the position of the emitter terminus. Positions of the emitters and spray voltages were adjusted to maximize detected level of the triply charged ion of angiotensin I (m/z ∼433) with 1 µM standard solution at the flow rate of 300 nL/min. Each full-scan mass spectrum was obtained

Figure 1. Schematic of the electrospray ionization system with inhouse-made carbon fiber emitter.

from four 300-ms microscans. Unless otherwise noted, all analyses were performed in infusion mode utilizing the LCQ integrated syringe pump without use of nebulization gas. Accumulated mass spectrometry data were processed with the Finnigan Xcalibur Qual Browser software package and exported to Microsoft Excel for evaluation. The ESI spray was observed with a CCD video camera using a 4× microscope objective. Monolithic Column Preparation. Monolithic columns were prepared as reported by Ro et al.20 Briefly, UV-transparent fluorinated hydrocarbon-coated silica capillary was rinsed with acetone and water, activated with 0.2 mol/L sodium hydroxide for 30 min, washed with water followed by 0.2 mol/L HCl for 30 min, washed with water and then acetone, and dried with a flow of helium. The capillaries were filled with a 30 vol % acetone solution of 3-(trimethoxylsilyl)propyl methacrylate, sealed, and left to react for 24 h at room temperature. The modified capillary was washed with acetone and dried. For the preparation of LMA/ EDMA column, the monomer mixture consisted of 180 mg of LMA and 120 mg of EDMA, porogenic solvents of 204 mg of 1-propanol and 61 mg of 1,4-butanediol, and 3 mg of DAP as an initiator. The polymerization solution was mixed ultrasonically into a homogeneous solution. The mixture was purged with helium for 3 min. A 30-cm-long capillary was attached to a syringe, filled with the solution and sealed with Teflon tubing. Location of the monolith was controlled with an aluminum foil mask. The length of the unmasked section of the capillary was kept constant at 10 cm. Polymerization was initiated by placing the capillary in a reaction chamber equipped with five 8-W UV lamps and irradiating for 20 min at 25 °C. After the polymerization was completed, the monolithic column was washed with methanol for 12 h using a HPLC pump to remove unreacted monomers and porogenic solvents. RESULTS Comparison of the CF Emitter and Commercial Nanotip Emitter. A simplified schematic for the electrospray ionization process is shown in Figure 1. The high voltage from the power supply is applied to a metal-coated CF emitter or nanoemitter, through which analyte solution flows. The front stainless steel union holds the emitter and contacts to high-voltage power supply. The other stainless steel union joins the emitter to the capillary tubing or monolithic column via a PEEK sleeve. (20) Ro, K. W.; Liu, J.; Busman, M.; Knapp, D. R. J. Chromatogr., A, submitted.

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Figure 2. Image of electrospray ionization using pointed carbon fiber emitter. Capillary i.d. was 75 µm; voltage applied was 3.5 kV; distance from tip to the counter electrode was 8 mm; the solution of MeOH/H2O/HOAc (48:48:4 by volume) was infused through the emitters at the flow rate of 400 nL/min.

The gold coating on the CF emitter was used for electric conduction of the ESI potential to the carbon fiber. The gold layer was covered by a perfluoralkyl film, to provide a hydrophobic character to the gold surface at the capillary exit. During ESI operation, it was observed that the base of the Taylor cone was confined to the inner diamter of the fused-silica capillary (see Figure 2). Using a blunt stainless steel emitter, operating with a high ESI voltage and a relatively high flow rate, multiple spray jets were observed and no normal Taylor cone could be seen.21 It has been speculated that the multiple points of electrospray nucleation were caused by the roughness resulting from sanding a metal tip.22 Others have argued that the multiple points of nucleation were not necessarily the result of sanding but just a self-adaptation of spray cones at high ESI voltage on a stainless steel surface.23 This phenomenon may relate to the random discharge and ejection at high voltage. The protruded CF emitter was designed to confine the nucleation at the sharp point and therefore generate a stable and controllable ESI process. As a result, while using a protruded pointed carbon fiber, we observed a stable and symmetric Taylor cone and ESI signals during operation in the voltage range from 1800 to 4500 V at the infusion flow rate from 0.1 to 5.0 µL/min (see Figure 3). Multiple points of origin for electrospray nucleation were not observed. The CF emitter shows good long-term and short-term stability for electrospray ionization (Figure 4). The RSD of the ion current for the m/z 433 ions from the CF emitter was 3.2% for long-term stability (1 h) and 2.9% for short-term stability (5 min). There is not a significant difference between long-term stability and shortterm stability. It indicates that CF emitter is a robust emitter, suitable for long-term ESI applications, such as interfacing with low flow rate chromatography. Similar operation of a conventional (21) Cloupeau, M.; Prunet-Foch, B. J. J. Aerosol Sci. 1994, 25, 1021. (22) Guzzetta, A. W.; Thakur, R. A.; Mylchreest, I. C. Rapid Commun. Mass Spectrom. 2002, 16, 2067-2072. (23) Shui, W.; Yu, Y.; Xu, X.; Huang, Z.; Xu, G.; Yang, P. Rapid Commun. Mass Spectrom. 2003, 17, 1541-1547.

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Figure 3. Voltage and flow rate effects on the ESI ion intensity. Plot A shows the relationship between voltage and ESI ion intensity (at the flow rate of 0.3 µL/min). Plot B shows the relationship between the flow rate and the ESI ion intensity (at the voltage of 2 kV). The distance from the emitter tip to the capillary was 3 mm.

nanospray emitter gave an RSD of 11% for long-term stability and 5.2% for short-term stability in the same period. The ESI conditions used for both emitters were those optimized for the New Objective Multilayer platinum-coated Pico Tip nanospray emitter (voltage, 2 kV; flow rate, 300 nL/min; tip to MS inlet distance, 3 mm). During nanospray emitter ESI operation, examination of long-term stabilities normally revealed an increase in the signal intensity in experiments running longer than 1 h. It appears that the carbon fiber and carbon ink are inert to the conditions of the ESI experiment. During the electrospray process, there were no matrix signals observed from the carbon fiber- or carbon ink-related material as shown in the mass spectrum of 1 pmol/µL angiotensin I from m/z 400 to 700. Compared to the conventional nanospray emitter, there were no new masses observed in the spectrum from the CF emitter (see Figure 5). Furthermore, when running a blank solution with the CF emitter, there was no specific mass observed in the spectrum above the noise level (spectrum not shown). Practical Properties for Sequencing Proteins and Identifying Peptides. A mixture of myoglobin and cytochrome c was digested as described above. To examine how many abundant peptides existed in the tryptic-digested protein mixture, the digest was diluted to 1 pmol/µL. Full-scan MS and data-dependent MS/ MS analyses were performed. The analyte solution was infused with a syringe pump at a flow rate of 200 nL/min. Full-scan and MS/MS data were collected for 1 min. Data-dependent MS/MS spectra for the 14 highest peptide signals were collected. The fullscan mass spectrum is shown in Figure 6a. The peptides noted in this m/z range are listed in Table 1. Upon processing of the acquired data with the Sequest program, most of the 11 detected peptides had high Xcorr scores (above 3.0). For peptides with multiple charged forms, the most abundant charge state is listed. The digest solution was diluted to 10 fmol/µL with a solution of MeOH/H2O/HOAc (48:48:4 by volume), and MS and data-

Figure 4. Comparison of the stabilities of the electrospray ionization signals between carbon fiber emitter and the New Objective 8-µm nanospray emitter. Angiotensin I, dissolved in the solution of MeOH/H2O/HOAc (48:48:4 by volume) was infused through the emitters at the flow rate of 300 nL/min. The ESI voltage was 2.0 kV.

Figure 5. Comparison of the characteristics of the electrospray ionization signals between carbon fiber emitter and the New Objective 8-µm nanospray emitter. Angiotensin I, dissolved in the solution of MeOH/H2O/HOAc (48:48:4 by volume) was infused through the emitters at the flow rate of 300 nL/min. The ESI voltage was 2.0 kV.

dependent MS/MS analyses were repeated on the dilute solution at the same flow rate of 200 nL/min. MS and MS/MS data were collected for 0.45 min. The data-dependent MS/MS spectra of the 10 highest peptide signals were collected. The full-scan mass spectrum is shown in Figure 6b. Detected peptides are listed in Table 1. Upon processing the data file with the Sequest software, many of the eight detected peptides were given Xcorr scores above 3.0. The eight detected peptides cover 41.2% of myoglobin and 44.2% of cytochrome c sequences, respectively. The Sequest software correctly indicated that the digest mixture contained peptides from both horse myoglobin and horse cytochrome c. The quantity of analyte consumed for identifying these two proteins is 0.9 fmol (10 fmol/µL × 200 nL/min × 0.45 min). Identification of each peptide requires 0.04 min. The absolute sample consumption is less than 0.1 fmol per peptide during the time each peptide is being analyzed. These results demonstrate that the CF emitter facilitates experiments leading to the identification of low levels of proteins. Use for LC/ESI-MS with Monolithic Capillary Columns. To test the adaptability of the emitter to chromatographic separations, the CF emitter was interfaced to a small inner diameter monolithic column.20 An aliquot of 0.2 µg of BSA tryptic digest was injected into the in-house-constructed monolithic column. A gradient elution of 4-70% acetonitrile was employed

to separate a BSA tryptic digest. Such a gradient requires robust operation of the emitter during changes in physical properties of the eluting solvent (dielectric properties, surface tension, etc.). Extremely stable electrospray ionization was achieved throughout the chromatographic separation. Twenty-nine peptides (of 56 predicted tryptic fragments of BSA) were separated and detected by LC/MS (see Figure 7). The detected peptides provided 57.7% sequence coverage for BSA. DISCUSSION A stable electrospray relies on suitable infusion flow rate and the ESI voltage. Jackson and Enke24 reported observation of a stable Taylor cone and an orderly generation of charged droplets only in the relatively constant current region. In the higher current region, multiple cones and jets appear and the droplet ejection is in rapidly changing off-axis directions. In the low-voltage, lowcurrent region, the Taylor cone forms briefly and ejects a pulsed droplet stream.24 Competition between surface tension and the Coulomb force controls the electrospray performance. In addition, the wetting property of the ESI emitter material is an important factor because the size of a spray droplet is dictated not only by the surface area of an emitter tip but also by the wetting properties (24) Jackson, G. S.; Enke, C. G. Anal. Chem. 1999, 71, 3777-3784.

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Figure 6. Full-scan mass spectra (m/z, 600-1500) of the digest of horse myoglobin and horse cytochrome c mixture. (a) The digest contained 1 pmol/µL peptides. (b) The digest contained 10 fmol/µL peptides. Sample solution flow rate was 200 nL/min. ESI voltage was 2.5 kV. Table 1. Peptides Found in Horse Myoglobin and Cytochrome c Digest original protein myoglobin

cytochrome c

MH+

obs, m/z 1.0 pmol/µL

Xcorr

1817.0 1607.8 1272.4 1379.7 1636.0 1886.2 1635.0 1599.7 1471.6 2210.6 780.0

909.8 804.1 636.6 690.3 818.5 629.5 817.7 801.0 736.2 737.5 779.6

3.6456 3.0067 3.0564 4.6886 1.2578 3.1455 2.6870 1.4412 0.9738 4.7409 1.0471

of the spray solution on the emitter material.23 In the multiple jet mode, multiple points of electrospray nucleation are initiated at the surface of a sanded tip.22 Generation of multiple spray cones at high ESI voltage on a stainless steel surface23 may relate to 3604 Analytical Chemistry, Vol. 76, No. 13, July 1, 2004

obs, m/z 10 fmol/µL

Xcorr

sequence

908.7 805.1

3.4028 2.6420

690.2 818.8 629.5 817.7

3.4925 0.7153 4.0139 1.7705

736.0 737.7

1.2485 3.4140

GLSDGEWQQVLNVWGK VEADIAGHGQEVLIR LFTGHPETLEK HGTVVLTALGGILK HGTVVLTALGGILKKK YLEFISDAIIHVLHSK IFVQKCAQCHTVEK KTGQAPGFTYTDANK TGQAPGFTYTDANK GITWKEETLMEYLENPKK MIFAGIK

the random/uncontrollable discharge at high voltage. Under the robust discharge conditions, droplet ejection from one Taylor cone is apparently insufficient to reach the charge separation equilibrium. Raising ESI voltage accelerates the discharge speed, which

Figure 7. Base peak chromatogram from the LC/ESI-MS analysis of tryptic digested BSA on a LMA/EDMA monolithic column (100-µm i.d., 375-µm o.d., 300 mm long (effective length, 100 mm)). Sample: 0.2 µg of tryptic-digested BSA. Mobile phase: (A) 3% acetonitrile in 0.15% aqueous TFA solution; (B) 70% acetonitrile in 0.15% aqueous TFA solution. Gradient elution program: 0 f 5 f 65 min, 3% f 3% f 100% B. Flow rate in column: 300 nL/min. Voltage for ESI with the carbon fiber emitter: +2.5 kV. MS detection: 400-2000 m/z.

is higher than the speed of liquid ejection from a single Taylor cone. Then the ejection by point discharge loses its selectivity and forms multiple spray jets at the blunt tip. We designed the protruding CF emitter to guide or confine the ESI process, so that a stable Taylor cone is present during operation. As discussed above, the CF emitter is tolerant to the variation in ESI conditions. The Taylor cone steadily envelops the carbon fiber tip and generates a smooth charge separation, even when sample infusion flow rate changes in a wide range from 0.1 to 5.0 µL/min. Lower flow rates were not tried, because the features of “true” nano-ESI are no longer present under these conditions.25 In this work, the infusion flow rate is controlled by the pumping speed. A benefit of the CF emitter is that the electric contact area between liquid and conductive tip is much larger than the conventional nanospray emitters. That means, to achieve the same electrospray ionization efficiency, the small size of the CF emitter aperture is not as critical as with a nanospray emitter. The larger aperture size of the CF emitter reduces the risk of clogging and facilitates use as a reliable emitter for low flow rate and highly sensitive electrospray ionization. For an electrode of fixed area, the charge-transfer process is rapid relative to the rate of diffusion. In the case of ESI, the portion of the metal-solution contact area at which oxidation occurs may increase back from the area closest to the tip as the current increases.26 This indicates that an increased electrode area can increase the current. The analyte solution contact area (effective electrode area) of the carbon fiber tip, at least the carbon cone part, is larger than a normal nanoemitter (a ring edge). This may help enhance the discharge efficiency. Erosion of gold coatings around the sharpened exit is an important issue in the operation of nanospray emitters. The CF emitter was operated for long periods without degradation of the gold coating. In the CF emitter, the contact area between analyte solution and the conductive surface is increased. Consequently, (25) Schmidt, A.; Karas, M.; Dulcks, T. J. Am. Soc. Mass Spectrom. 2003, 14, 492-500. (26) Van Berkel, G. J.; Giles, G. E.; Bullock, J. S., IV.; Wendel, M. W.; Gray, L. J. Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, ASMS: Santa Fe, NM, 1998; p 132.

the efficiency of direct heterogeneous electron-transfer reactions and the electrochemical oxidation rate are increased. As a result, the charge separation is enhanced. While the carbon fiber may be eroded by the electrochemical oxidation during ESI, it does not significantly change the emitter surface area and properties, thereafter the discharge conditions. We believe that is why we observe long lifetimes for the CF emitters. Generally, it was observed that ESI operation with a CF emitter could last 15-20 h without significant signal intensity change. In comparison, conventional nanospray emitters have shorter lifetimes than the CF emitters. Utilizing the same experimental conditions as the CF emitter, the lifetime of the compared commercial nanospray emitter was ∼10 h. A significant loss of the gold coatings was observed around the nanospray emitter aperture. Similarly, arcing also can damage the gold coatings around the nanospray tip. The rate of charge separation is a function of the rate of influx of analyte, not the field strength.24 For a fixed current, a smaller electrode area means a higher current density.27 The current in the gap is determined by the rate of charge separation at the emitter tip. It proportionally relates to the ESI efficiency. To the same ESI efficiency, a larger contact area between conductive surfaces to the sample solution leads to lower current density. This may prolong the lifetime of an ESI emitter. The CF emitter has a larger contact area, which should be favorable for extending its lifetime. Improper positioning of the CF emitter may cause corona discharge, arcing, or both between the emitter and MS interface. Arcing can damage the electric connection between the gold (the connection for the high-voltage power supply) and carbon fiber. In this case, a recoating of the gold film is necessary to reestablish the electrical connection between carbon fiber tip and the outer surface of the fused-silica capillary. To avoid the formation of corona, one can position the emitter farther away from the MS sampling capillary. After application of high voltage, the emitter can be moved nearer to the MS interface for smooth ESI operation. (27) Van Berkel, G. J. J. Am. Soc. Mass. Spectrom. 2000, 11, 951-960.

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The CF emitter is well matched to the chromatography conditions of 100-µm-i.d. capillary monolithic columns. The mobilephase flow rate of 300 nL/min falls into the optimized infusion flow rate range of CF emitters. The liquid consumption range of the CF emitter (0.1-5.0 µL/min) is compatible with the general flow rates of nanoliquid chromatography techniques. Generally, a stable ESI process requires that the sample solvent has a suitable surface tension. For example, it is difficult to get a stable spray in water solution without adding any methanol. The carbon fiber emitter helps to eliminate the unstable ESI problem, which may arise from the surface tension and dielectric properties variation due to changes in the organic component in the elute, especially at low to medium ESI voltage. CONCLUSIONS A pointed carbon fiber ESI emitter has been developed for nanoliquid sampling. The protruded sharp carbon fiber guides an extremely stable charge separation at the emitter tip point. The hydrophobic surface surrounding the fiber restricts the Taylor cone bottom to the inner diameter of the exit. This small Taylor cone improves the electrospray performance, thereby enhancing the detectability of ESI/MS. This emitter is able to generate stable electrospray in a wide range of flow rate, ESI voltage, and surface tension variations. The high stability of ESI operation, especially in terms of short-term stability, makes it possible to analyze a peptide mixture over a short period while collecting reliable MS

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and MS/MS data. The sensitivity of the CF emitter is comparable to that of a conventional nanospray emitter. Such performance is helpful for obtaining data-dependent sequencing information from low concentration level peptide solutions. The durable and sensitive CF emitter has been used for identifying 1-fmol quantities of peptides in a complicated peptide mixture. Eight out of 11 observed peptides were identified with high Sequest Xcorr scores, facilitating identification of the parent proteins in the mixture. In addition, CF emitter operation was shown to be sufficiently robust for interfacing to 300 nL/min flow rate chromatography to mass spectrometry without significant loss of the LC resolution. ACKNOWLEDGMENT This work was supported in part by NIH Grant CA86285, the NHLBI Proteomics Initiative via Contract N01-HV-28181, and the Postdoctoral Fellowship Program (to K.W.R.) of Korean Science and Engineering Foundation (KOSEF). We thank Lauren Magaldi for editorial assistance. This work was carried out in the MUSC Biomolecular Mass Spectrometry Facility.

Received for review December 17, 2003. Accepted April 8, 2004. AC030419I