A Sheathless Nanoflow Electrospray Interface for On-Line Capillary

Anal. Chem. 2003, 75, 1615-1619

A Sheathless Nanoflow Electrospray Interface for On-Line Capillary Electrophoresis Mass Spectrometry George M. Janini,* Thomas P. Conrads, Kenneth L. Wilkens, Haleem J. Issaq, and Timothy D. Veenstra

Analytical Chemistry Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702

A novel, rugged capillary electrophoresis-electrospray ionization (CE-ESI) interface where the separation column, an electrical porous junction, and the spray tip are integrated on a single piece of a fused-silica capillary is described. ESI is accomplished by applying an electrical potential through an easily prepared porous junction across a 3-4-mm length of fused silica. A stable electrospray is produced at nanoflow rates generated in the capillary by electrophoretic and electroosmotic forces. The interface is particularly well suited for the detection of lowfemtomole levels of proteins and peptides. The ruggedness of this interface was evident by the continuous operation of the same column for over a 2-week period with no detectable deterioration in separation or electrospray performance. The new interface was used for the LC-ESI-MS separation and analysis of peptides and proteins. Injection of 25 fmol of [Glu1]-fibrinopeptide B using the new device produced a CE-ESI-MS electropherogram with a signal-to-noise ratio of over 100 for this peptide. Mass spectrometry (MS) is arguably among the most useful detection schemes for capillary electrophoresis (CE) and liquid chromatography (LC), largely due to the fact that other common detection techniques (e.g., UV, visible, and fluorescence) provide only limited information about the analytes’ identities, particularly in the case of peptides and proteins. CE has been utilized for the separation of analytes prior to analysis by both fast atom bombardment1 and matrix-assisted laser desorption/ionization MS instrumentation,2 but by far the best compatibility is obtained by interfacing CE directly on-line with electrospray ionization (ESI) MS. The successful operation of a CE-ESI-MS system requires a closed circuit for both the CE separation and the electrospray processes. Three major designs that satisfy this requirement have been advanced, namely, coaxial sheath flow,3 liquid junction,4 and sheathless flow.5 Coaxial sheath flow is the basis of most * Corresponding author. Phone: 301-846-7189. Fax: 301-846-6037. E-mail: [email protected]. (1) Moseley, M. A.; Deterding, L. J.; Tomer, K. B.; Jorgenson, J. W. J. Chromatogr. 1989, 480, 197. (2) Preisler, J.; Foret, F.; Karger, B. L. Anal. Chem. 1998, 70, 5278. (3) Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988, 60, 436. 10.1021/ac020661+ CCC: $25.00 Published on Web 02/25/2003

© 2003 American Chemical Society

commercial instruments, though it suffers in sensitivity. This arises largely from the relatively high sheath flow compared to the flow from the CE capillary, resulting in not only a large dilution of the eluting analytes but also in hindered desorption of ions due to the nonoptimal electrospray that results at such high flow rates. Coupling CE on-line with MS through a liquid junction requires tedious capillary alignment and end-to-end butting of the separation capillary and the spray tip. Poor alignment compromises sensitivity and separation efficiency by spreading of sample in the relatively large dead volume of the liquid junction. In principle, the sheathless interface is the most optimal design for coupling CE on-line with ESI-MS because of the compatible flow rates of each technique, which leads to high desorption and ionization efficiencies and, consequently, high sensitivity. In contrast to the sheath flow interface, analytes are not diluted and no additional fluids are present at the spray tip other than the CE column effluent. A variety of sheathless designs have been described that satisfy the requirement of closing the CE separation capillary circuit while simultaneously providing an electrical potential to the spray tip. These, for example, include the use of a single capillary whereby electrical contact is established through the following: (a) coating the capillary outlet with a conductive metal5-12 or polymer;13 (b) insertion of a conductive wire into the outlet of the capillary14 or through a small pinhole in the wall of the capillary;15,16 (c) splitting the capillary effluent at or near the capillary outlet to fill the gap between the capillary and an outer coaxial metallic sleeve;17-19 or (d) adjusting the position of the (4) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R. Biomed. Environ. Mass Spectrom. 1989, 18, 844. (5) Olivares, J. A.; Nguyen, N. T.; Yonker, C. R.; Smith, R. D. Anal. Chem. 1987, 59, 1230. (6) Kelly, J. F.; Ramaley, L.; Thibault, P. Anal. Chem. 1997, 69, 51. (7) Barraso, M. B.; deJong, A. P. J. Am. Soc. Mass Spectrom. 1999, 10, 1271. (8) Samskok, J.; Wetterhall, M.; Jacobsson, S.; Markides, K. J. Mass Spectrom. 2000, 35, 919. (9) Chang, Y. Z.; Her, G. R. Anal. Chem. 2000, 72, 626. (10) Figeys, D.; Oostveen, I.; Ducert, A.; Aebersold, R. Anal. Chem. 1996, 68, 1822. (11) Kriger, M. S.; Cook, K. D.; Ramsey, R. S. Anal. Chem. 1995, 67, 385 (12) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1. (13) Maziarz, E. P.; Lorentz, S. A.; White, T. P.; Wood, T. D. J. Am. Soc. Mass Spectrom. 2000, 11, 659. (14) Fang, L.; Zhang, R.; Williams, E. R.; Zare, R. N. Anal. Chem. 1994, 66, 3696. (15) Cao, P.; Moini, M. J. Am. Soc. Mass Spectrom. 1998, 9, 1081. (16) Smith, A. D.; Moini, M. Anal. Chem. 2001, 73, 240. (17) Moini, M. Anal. Chem. 2001, 73, 3497.

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Figure 1. Schematic diagram of the sheathless CE-ESI-MS interface.

outlet of the capillary such that electrical contact is established through the air to the grounded inlet capillary of the MS.20 Another strategy for the fabrication of a sheathless interface is to use two pieces of capillary whereby the CE capillary is connected to a short spray tip via a sleeve. The sleeve could be a piece of microdialysis tubing,21 stainless steel tubing,22 or a micro-tee.23 A three-piece design whereby electrical contact is established through a porous glass joint that is butt connected, using Teflon sleeves, to the CE column from one side and the spray tip from the other has also been reported.24 Although these approaches do produce operational interfaces, their fabrication requires delicate manipulation of miniaturized components and they suffer in their robustness. In the two-piece approach, the CE-separated zones are invariably broadened at the junction between the separation column and the tip, since the inside diameter of the sleeve has to be larger than the outside diameter of the separation capillary. Furthermore, these junctions often suffer from misalignment and imperfect butting of the two pieces of capillary. Single-capillary methods appear to disrupt the CE separation the least; however, metal coatings on fused-silica capillaries are not durable and drilling pinholes through capillary walls is a delicate and irreproducible procedure. Once operational, a split-flow interface is highly sensitive, as demonstrated by Moini et al., who reported the separation and detection of proteins from human red blood cells at attomole levels.18 Sheathless interfacing has been the subject of several recent reviews, where the advantages and limitations of this design have been enumerated.25-28 In this paper, we present a rugged and simple device to couple CE on-line with MS where the separation column, a porous electrical junction, and the electrospray tip are integrated on a single piece of a fused-silica capillary. The key to this design is that electrical contact for the low potential side of the CE and for the electrospray tip is provided by a porous junction created by hydrofluoric acid etching through a 3-4-mm length of the capillary (18) Moini, M.; Demars, S. M.; Huang, H. Anal. Chem. 2002, 74, 3772. (19) Petersson, M. A.; Hulthe, G.; Fogelqvist, E. J. Chromatogr., A 1999, 854, 141. (20) Mazereeuw, M.; Hofte, A. J. P.; Tjaden, U. R.; van der Greef, J. Rapid Commun. Mass Spectrom. 1997, 11, 981. (21) Severs, J. C.; Smith, R. D. Anal. Chem. 1997, 69, 2154. (22) Figeys, D.; Ducret, A.; Yates, J. R.; Aebersold, R. Nature Biotechnol. 1996, 14, 1579. (23) Tong, W.; Link, A.; Eng, J. K.; Yates, J. R. Anal. Chem. 1999, 71, 2270. (24) Settlage, R. E.; Russo, P. S.; Shabanowitz, J.; Hunt, D. F. J. Microcolumn Sep. 1998, 10, 281. (25) Tong, W.; Yates, J. R. Chromatographia 2001, 53 (Suppl), S90. (26) Ding, J.; Vouros, P. Anal. Chem. 1999, 71, 378A. (27) Gelpi, E. J. Mass Spectrom. 2002, 37, 241. (28) Moini, M. Anal. Bioanal. Chem. 2002, 373, 466.

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wall near the outlet (i.e., spray tip) end. This device is easily mounted on any MS system equipped with an ESI interface, and switching from LC to CE is as simple as removing the LC column and mounting the device in its place on the ESI assembly. EXPERIMENTAL SECTION Interface Fabrication. Fused-silica capillaries (Polymicro Technologies, Phoenix, AZ) were used to fabricate the sheathless interfaces according to the following procedure: Spray tips were made by applying heat from a microtorch while pulling gently. The resulting long tapered tip is later trimmed to the desired tip inside diameter using a glass tube cutter. The polyimide coating was removed from a 3-4-mm section of the capillary at a distance of 5 cm from the spray tip, end and the capillary was trimmed to a total length of 60 cm. The capillary was mounted on the porous junction assembly as shown in Figure 1. The assembly was constructed from a 4.5 cm × 1.5 cm Plexiglas slide, where a groove was milled along its length having a depth slightly larger than the capillary’s outside diameter, and a 1.5 cm × 1 cm i.d. Plexiglas reservoir that is glued on top of the slide. The fused-silica capillary was threaded in the groove from one side and out the other side of the reservoir to position the exposed section of the fused-silica capillary inside the reservoir. 5 Minute Epoxy glue (Devcon, Riviera Beach, CA), was applied to the outside of the reservoir around the capillary to seal the two holes and to pin the capillary on the slide. HF etching of the exposed fused-silica segment was conducted according to a procedure first reported by Hu et al.29 and later utilized by Settlage et al.24 and by Wei and Yeung,30 with modifications. The reservoir was filled with 20% HF to cover the exposed fused-silica section and allowed to incubate in a hood for 5 h at room temperature. The capillary was continuously monitored, and the etching reaction was terminated as soon as electrical conductance was established through the already porous capillary wall. Over the course of the reaction (∼6 h), the capillary wall thins to about 15-20 µm. Although this porous junction created by HF etching is fragile, it is durable since it is firmly held inside the reservoir, which also serves as the buffer reservoir for closing the CE circuit and providing the spray voltage. CE-MS. An ion trap mass spectrometer (LCQ-DECA XP, ThermoFinnigan, San Jose, CA) equipped with a nanoelectrospray ionization source was used for all CE-MS experiments. The porous junction assembly was mounted on the nanoelectrospray source as shown in Figure 2, and fine positioning of the spray tip was achieved by using the manufacturer’s XYZ stage. The (29) Hu, S.; Wang, Z.-L.; Li, P.-B.; Cheng, J. K. Anal. Chem. 1997, 69, 264. (30) Wei, W.; Yeung, E. S. Anal. Chem. 2002, 74, 3899.

Figure 2. CE-ESI-MS interface mounted on the LCQ-DECA MS nanoelectrospray assembly.

reservoir was filled with the CE running electrolyte, and the spray voltage was supplied by a platinum electrode immersed in the electrolyte from an external power supply (Series EL, Glassman High Voltage Inc., High Bridge, NJ 08829). For MS analysis, the spray voltage was adjusted between 2.0 and 2.5 kV for optimum spray stability, and a capillary temperature of 180 °C was used for all experiments. The instrument was operated in a datadependent tandem MS mode in which each full-scan mass spectrum was followed by a tandem MS scan of the most intense ion observed in the previous scan. Normalized collision energy was set to 38%. A P/ACE System MDQ CE instrument was used to conduct the CE separations (Beckman Coulter, Fullerton, CA). The MDQ instrument was configured to accept a windowless capillary cartridge, where the inlet side of the capillary was threaded into the detector end of the cartridge and the spray tip end extended to the outside of the instrument. Both the MS and the CE instruments were controlled using ThermoFinnigan Xcalibur software (San Jose, CA). Chemicals. Formic acid and acetic acid were obtained from Fluka Chemical Corp. (Milwaukee, WI), 48% HF was obtained from Aldrich (St. Louis, MO), acetonitrile (HPLC grade) was purchased from EM Science, Merck (Darmstadt, Germany), and 5 Minute Epoxy was obtained from Devcon. [Glu1]-fibrinopeptide B, trypsin, equine apomyoglobin, and the bioactive peptide mixture were purchased from Sigma (St. Louis, MO). Water for all uses was doubly distilled and deionized using a NANOpure diamond water system (Barnstead Internations, Dubuque, IA). Protein Digestion. A total of 60 nmol of apomyoglobin was dissolved in 1 mL of 100 mM NH4HCO3, pH 8.2, and digested with trypsin for 18 h at 37 °C, at a protein/trypsin ratio of 50:1 (w/w). The digest was lyophilized to dryness and resuspended in 1 mL of 10 mM HCl. RESULTS AND DISCUSSION A simple device was developed for the direct on-line coupling of CE with MS that provides for the highly sensitive analysis of

peptides and proteins. The device offers advantages over existing CE-MS interfaces, including ease of fabrication, ruggedness, durability, and a true zero dead volume junction between the separation column and the spray tip. One aspect of CE that makes it particularly suited for high-throughput proteomic investigations is the ability to conduct fast and efficient separations of peptides and protein digests, based upon differences in analyte charge-tosize ratios. CE is well known for its highly selective separations even though the peak capacity is less than that of LC. In addition, because the CE capillary is constructed using open tubular fused silica, there is minimal opportunity for contamination due to carryover effects from separation to separation. Since there is no need to reequilibrate the column as in LC, the time between consecutive CE experiments is much less than in the case of LC-based separations with gradient elution. The present design of the sheathless CE-MS interface is illustrated in Figure 1. The key to conducting both the CE separation and the ESI is the construction of a porous junction by etching an exposed capillary segment with HF, which results in the thinning of the capillary wall enough to generate electrical contact without affecting the inside diameter of the capillary. The porous junction is fragile, but once it was made and protected inside the reservoir it proved to be durable. Throughout our investigation we have fabricated several of these CE columns and each lasted for more than 2 weeks (e.g., ∼100 CE-MS runs) without any observable deterioration in either CE separation or electrospray performance. To demonstrate the sensitivity of this on-line sheathless CE design, we analyzed a series of peptide mixtures. The electropherogram of 25 fmol of a standard peptide ([Glu1]-fibrinopeptide B) detected on-line by an ion trap mass spectrometer is shown in Figure 3A. The [M+2H]2+ [Glu1]-fibrinopeptide B molecular ion (m/z 786.8) shown in Figure 3B demonstrates the high signalto-noise ratio (S/N > 100) typically observed in these experiments conducted using the present sheathless on-line CE design. The Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

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Figure 4. CE-ESI-tandem MS of a mixture of bioactive peptides. Experimental conditions: as in Figure 3. Sample concentration, 3 µg/ mL; injection time, 10 s at 0.5 psi; solutes, (1) bradykinin; (2) substance P; (3) bradykinin fragments 1-5; (4) [arg]-vasopressin; (5) luteinizing hormone releasing hormone; (6) bombesin; (7) leucine enkephalin; (8) methionine enkephalin; (9) oxytocin. Inset: corresponding CE separation of the same peptide mixture at a concentration of 25 µg/mL.

Figure 3. CE-ESI-tandem MS of 25 fmol of [Glu1]-fibrinopeptide B: (A) base peak electropherogram; full-scan mass spectrum of the [M + 2H]2+ [Glu1]-fibrinopeptide B molecular ion; (C) tandem MS fragment spectrum of the m/z 786.8 ion. Conditions: column, bare fused-silica capillary, 60 cm × 360 µm o.d. × 75 µm i.d. × 25 µm tip i.d.; separation potential, 15 kV; observed CE current, 16 µA; running electrolyte, 1 M acetic acid, pH 2.4; temperature, 22 °C; sample concentration, 2 µM; injection time, 5 s at 0.5 psi (∼12.5 nL total injection volume). Other experimental conditions, as in the Experimental Section.

interface operates under nanoflow conditions, which results in higher S/N. Based on the observed S/N, it is estimated that the lower sensitivity limit is ∼900 amol (assuming a detection limit at a S/N ) 5). This represents a ∼100-fold improvement in detection limit relative to analysis of the same sample on an equivalent CE column employing a sheath flow interface (results not shown). The viability of using this sheath less CE design with tandem MS for the identification of peptides from the resulting fragment ion spectrum is illustrated by Figure 3C. Information from this spectrum was used to search a nonredundant protein database (http://www.ncbi.nih.gov) using the program SEQUEST, which resulted in the positive identification of [Glu1]-fibrinopeptide B with a Xcorr score of 4.8 (data not shown). The separation of a mixture of nine bioactive peptides at a concentration of 3 µg/mL (injection, 75 fmol of each peptide) is shown in Figure 4. The inset to this figure shows the corresponding separation of the same bioactive peptide mixture at a concentration of 25 µg/mL, using similar CE conditions with UV detection at 214 nm. Compared to UV detection, the relative peak heights vary considerably because of differences in the ionization 1618 Analytical Chemistry, Vol. 75, No. 7, April 1, 2003

efficiency of different peptides. Also MS-generated peaks are broader, most likely, due to lack of temperature control in the CE-MS setup and the slow rate of data acquisition by the MS. The direct detection of MS, however, allowed all of the peptides within this mixture to be easily identified based on their MS spectra. The CE-MS analysis of a tryptic digest of apomyoglobin is shown in Figure 5. In this analysis 100 fmol of the digest was injected onto the CE column and the peptides were detected in a data-dependent MS/MS mode. The electropherogram for this tryptic digest is shown in Figure 5A. The MS spectrum of the peptide highlighted with an arrow is shown in Figure 5B. The singly and doubly charged species of this peptide were detected with very high S/N, particularly for the doubly charged ion. The tandem MS spectrum of the doubly charged parent ion is shown in Figure 5C. Analysis of this spectrum using SEQUEST identified this peptide as HGTVVLTALGGILK from apomyoglobin with an Xcorr score of 4.95. Indeed, an almost complete y ion series was identifiable for this peptide from the resulting tandem MS spectrum. Table 1 lists peptides that were identified by tandem MS with high Xcorr, and Table 2 gives a list of all tryptic peptides that were identified by matching the molar mass of ions from the MS spectra with a list of expected peptides generated from the putative protein sequence. CE is particularly well suited for the separation of peptides and proteins in acidic buffers as CE columns are known to be stable over extended periods of time when operated under these conditions. Formic acid and acetic acid were tested as running electrolytes with and without the addition of organic solvents, and the best performance in terms of CE separations and MS sensitivity was obtained with 1 M acetic acid (pH 2.4) without

Table 2. Identified Peptides from CE-MS Peptide Mapping of Horse Apomyoglobin Tryptic Digest

Figure 5. CE-ESI-MS analysis of 100 fmol of a tryptic digest of horse apomyoglobin. Experimental conditions: as in Figure 3. (A) Base peak electropherogram; (B) molecular ion scan of the [M + 2H]2+ ion at m/z ) 690.7; (C) CID fragment ion spectrum of the [M + 2H]2+ ion at m/z ) 690.7. The CID spectrum was searched against the NCBI nonredundant database using SEQUEST and identified as HGTVVLTALGGILK with an Xcorr ) 4.95. Table 1. Identified Peptides from CE-MS/MS Analysis of Horse Apomyoglobin Tryptic Digest peptide

sequence

Xcorr

fragment ions

1 2 3 4 5

HLKTEAEMK YLEFISDAIIHVLHSK HGTVVLTALGGILK YKELGFQG GLSDGEWQQVLNVWGK

2.95 4.97 4.95 2.66 4.63

14/16 22/30 23/26 12/14 21/30

any additives. This electrolyte appears to be the most ideal for this application because it produced steady electrospray and lowfemtomole sensitivity without the use of organic solvent additives. Formic acid, on the other hand, resulted in higher current and less sensitive MS detection. At pH 2.4, all peptides are positively charged and move toward the cathode at the spray tip outlet. The flow rate through the column is generated by electroosmotic flow with some assistance from the electrospray process. Electroosmotic flow is very low under acidic conditions and cannot be easily measured by CE-MS because neutral electroosmotic flow markers do not generate a signal; however, an estimate of the upper limit of the flow rate can be easily obtained from Figure 4. Oxytocin, the last eluting peptide in the figure, took over 26 min

fragment

sequence

residues

1 2 3 4 5 6 7 8 9 10 11 12

GLSDGEWQQVLNVWGK VEADIAGHGQEVLIR FDKFK HLKTEAEMK HGTVVLTALGGILK GHHEAELK PLAQSHATK YLEFISDAIIHVLHSK HPGNFGADAQGAMTK ALELFR NDIAAK YKELGFQG

1-16 17-31 43-47 48-56 63-76 79-86 87-95 102-117 118-132 133-138 139-144 145-153

to migrate a distance of 60 cm with a linear velocity of 2.3 cm/ min. This linear velocity translates to a volumetric flow rate of ∼100 nL/min for a 75-µm-i.d. column. This flow rate represents an upper limit because oxytocin migrates under electrophoretic as well as electroosmotic forces, and electroosmotic flow may be much less than this upper limit. The addition of organic solvents (acetonitrile or methanol), although beneficial to the electrospray process, especially with wide spray tips (>30 µm), caused peak broadening and deterioration in the separation of peptides. Adjustment of pH with ammonia or triethylamine caused the formation of dominant peptide adducts. During the course of this investigation, we have used 1 M acetic acid and obtained stable electrospray with 75- and 50µm-i.d. columns having spray tip diameters ranging from 8 to 50 µm. Small-diameter tips (