Corresfiondence Anal. Chem. 1995, 67, 462-465
Direct Electrospray Ion Current Monitoring Detection and Its Use with On-Line Capillary Electrophoresis Mass Spectrometry Jon H. Wahl, Steven A. Hofstadler, and Richard D. Smith* Chemical Sciences Department, Pacific Northwest Laboratory, Richland, Washington 99352
A novel detection scheme for use with the capillary electrophoresis-electrospray ionization interface is presented, based upon the direct measurement of electrospray ion current after expansion into vacuum, that is shown to provide the basis for a simple detector or for simultaneous dual detectionwith mass spectrometry. The utility of this novel dual-detectionscheme is illustrated with capillary electrophoresis Fourier transform ion cyclotron resonance (FI'ICR) mass spectrometry in which spectra are acquired only when a solute zone elutes from the CE capillary. This acquisition scheme affords significantly improved sensitivity and permits the acquisition of longer time domain signals without sacrificing separation efficiency. Mass resolving power in excess of lo5 (hhm) is demonstrated by FI'ICR for components from a peptide/protein mixture comprised of analytes with molecular masses ranging from 2 to 29 kDa. The application of capillary electrophoresis (CE) in chemical and biochemical characterization is continuing rapid growth due to the speed, versatility, simplicity, and efficiency of the separations Moreover, the use of capillary electrophoresis with on-line mass spectrometry (MS) has also continued to to where most of the CE/MS results reported to date are based on the electrospray ionization (ESI) interfa~e.~ An advantage of the ESI interface is that very large molecular compounds can be detected at relatively modest m/z due to the multiple charging5 phenomenon inherent in the ESI process. Consequently, the necessary m/z range that must be obtained for such applications is signifmntly reduced compared to other ionization methods (most of which are also less effective for larger molecules). Extraordinary detection sensitivity is also possible using CE/MS; we have recently demonstrated sub femtomole detection limits for small proteins using small inner diameter capillaries and a low-resolution quadrupole mass spectrometer.6 Generally, CE (1) Engelhardt, H.; Beck, W.; Kohr, J.; Schmitt, T. Angew. Chem., Inf. Ed. Engl. 1993,32, 629-766. (2) Mesaros, J. M.; Ewing, A G.; Gavin, P. F. Anal. Chem. 1994,66,527A537.4. (3) Smith, R D.; Wahl, J. H.; Goodlett, D. R; Hofstadler, S. A Anal. Chem. 1993,65,A574A584. (4) Olivares, J. A; Nguyen, N. T.; Yonker, C. R; Smith, R D. Anal. Chem. 1987,59, 1230-1232. (5) Yamashita, M.; Fenn, J. B. J Phys. Chem. 1984,88,4451-4459.
462 Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
applications require analyte amounts in the femtomole range and above; with conventional quadrupole mass spectrometric detection resolution, scan range and/or scan speed (as well as resolution!) must be compromised to obtain these detection limits. The ESI Fourier transform ion cyclotron resonance (ESIFTICR) combination was pioneered by McLafferty, Hunt and coworkers, who initially demonstrated that high-resolution precision mass measurements can be obtained for proteins and peptides.? A unique advantage of FTICR mass spectrometry for the study of biopolymers is the ability for simultaneous detection over a wide m/z range. Furthermore, ultrahigh MS resolution with low ppm mass measurement error and the ability to perform higher order tandem MS methods (i.e., MSn,where n t 2) for structural studies are possible because of the nondestructive detection scheme. We have developed CE-ESI-FRMCR instrumentation incorporating features that allow rapid (i.e., < 1 s) manipulation of pressures in the FTICR cell between those that are optimum for ion trapping and cooling (i.e., > Torr) and those for highresolution detection (< Torr), facilitating spectral acquisition on a time scale compatible with most on-line separation techniquesS8A key feature of this instrumental configuration is the addition of a pair of electromechanical shutters which serve to block the conductance limits during noninjection events in order to provide enhanced differential pumping. In this work, we present an additional benefit of the electromechanical shutter assembly, the ability to monitor the ion current from the electrospray source in a manner that allows it to be used as a general monitoring scheme for large molecules, and demonstrate the basis for a simple general detector for use with separations. As will also be demonstrated, this ion current monitoring scheme serves as a postionization detector which, when used with on-line separations, also provides an accurate indication of the number of analyte ions entering the spectrometer (allowing a semiquantitative evaluation of the mass spectrometers trapping and detection capabilities) and provides an accurate indication of when the analytes are entering the spectrometer. While the utility of this ion current detection scheme is demonstrated in conjunction with (6) Wahl, J. H.; Goodlett, D. R; Udseth, H. R; Smith, R D. Anal. Chem. 1992, 64,3194-3196. (7) Henry, K D.; Williams, E. R; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. PYOC. Natl. Acad. Sci. U.S.A. 1989,86, 9075-9078. (8) Winger, B. E.; Hofstadler, S. A; Bruce, J. E.; Udseth, H. R; Smith, R D., J Am. SOC.Mass Spectrom. 1993,4,566-577. 0003-2700/95/0367-0462$9.00/0 0 1995 American Chemical Society
the CEFTICR interface, it should be broadly applicable to a family of interfaces which employ ESI to directly couple a solution phase separation, including CE or LC,to a mass spectrometric detection scheme. Preliminary studies indicate that this technique may provide adequate sensitivity to serve as a low-cost stand-alone detection scheme for analytes that are amenable to ionization by ESI. EXPERIMENTAL SECTION The CE instrument used for these experiments was built inhouse and consists of a Plexiglas box that isolates the high-voltage source and houses the sample and buffer vials. In these studies, surface-derivatized fused-silica capillaries (lo@ and 2@,m i.d., -15Ckpm o.d., and 1-m length) were utilized (Polymicro Technologies, Phoenix AZ). The inner walls of the capillaries were chemically modified with aminopropylsilane (APS)!,lo Approximately 1 cm of the polyimide coating was removed from the capillary terminus, and the exposed fused silica was etched with a 40% hydrofluoric acid solution (Aldrich, Milwaukee,WI) to taper the outlet of the analytical capillary. To electrospray directly from the tapered CE terminus, a gold conductive coating (Epoxy Technology,Billerica MA) was applied which establishes electrical contact at the CE capillary terminus to detine the CE electric field. A sheath gas of sulfur hexduoride around the capillary terminus is used to suppress corona discharge. An advantage of the conductive tip interface over a sheath-type ESI interface is that a significantly lower background signal is generated." The electrospray was produced by use of a f3.8 kV gradient between the CE capillary terminus and the heated desolvation inlet capillary to the first stage of the mass spectrometer vacuum system. The running electrolyte for all CE separations was a 0.01 M acetic acid solution, pH 3.4. As a result of the aminopropylsilane surface modification and the acidic buffer system used, the inner walls of the capillaries have a net positive charge, which results in an electroosmotic flow in the opposite direction compared to uncoated fused-silica capillaries. The electrical fields strengths used for these studies were --200 V/cm-'. The peptide/protein test mixture used in this study was composed of somatostatin (1638 Da), ubiquitin (8 565 Da), a-lactalbumin (14 175 Da), lysozyme (14 306 Da), myoglobin (16 951 Da), and carbonic anhydrase I (28 802 Da) (Sigma, St. Louis, MO), each at 50 pM in doubly distilled deionized water. The sample was electrokinetically injected at --38 V/cm, and amounts injected were estimated on the basis of the electromigration rates of the components. The Fourier transform ion cyclotron resonance mass spectrometer used in this work was described in considerable detail elsewhere! and only a brief description is given here. The spectrometer is based on a 7-T superconducting magnet with a &in. room-temperature bore. Ions are formed by electrospray ionization in a modhed Analytica (Branford, CT) ESI source equipped with a heated metal capillary inletI2 and are injected through the fringing magnetic field using two sets of radio frequency Q-only quadrupoles; each set of quadrupoles is preceded by a high-speed mechanical shutter.13 As shown in (9) Bruin, G. L. M.; Huiden, R; Kraak,J. C.; Poppe, H. ]. Chromafogr.1989, 480,339-343. (10) Lukacs,K D.Diss. Absfr. Inf. 1983,44, 3766. (11) Wahl, J. H.; Gale, D. C.; Smith,R D.]. Chromafogr. A 1994,659,217222. (12) Chowdhury, S. K; Katta,V.; Chait, B. T.Rapid Commun. Mass Specfrom. 1990,4, 81-87.
4 OPEN
CLOSED
Figure 1. Shutter assembly. (a) Each consists of a 3-mm conductance limit, which is obscured by a solenoid-driven wiper assembly. The conductance limit and wiper are electrically common and are electrically isolated from the vacuum chamber. (b) The first shutter (Sl)is positioned immediately adjacent to the ESI source while the second shutter (S2) separates the short (Ql) and long (Q2) sections of tf-only quadrupoles, which extend 55.6 cm to the trapped ion cell (not shown).
Figure la, each shutter assembly consists of a 3mm conductance limit which is obscured by a solenoid-driven "wiper". The shutters are gated open and the rf-only quadrupoles are engaged only during ion injection into the FTICR trap (typically 50-100 ms for each spectrum). During other pulse sequence events, the conductance limits are blocked by the wiper arms so as to enhance differential pumping. The conductance limits and wipers are electrically isolated from the rest of the vacuum chamber, which enables direct ion current measurement from the wiper(s) with a Keithley (Cleveland, OH) Model 617 programmable digital electrometer. The electrometer was coupled to a Linear chart recorder (Altech Associates, Deerfield, IL) to provide a dynamic record of the electrospray ion current leaving the ESI source (typically 1-100 PA). As shown in Figure lb, the k s t shutter is positioned immediately adjacent to the ESI source. A 10.2-cm set of rf-only quadrupoles separates the first and second shutters. By opening the shutters only during ion injection events, a lower gas load is admitted to the trapped ion cell during the majority of the pulse sequence. The lower base pressure thus obtained enables longer transient lifetimes, which in turn produces higher resolution mass measurements. A pressure reduction of > 12 orders of magnitude from the atmospheric pressure ion source to the trapped ion cell (base pressure
