Anal. Chem. 1998, 70, 3235-3241
Capillary Isoelectric Focusing-Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Protein Characterization Liyu Yang and Cheng S. Lee*,†
Department of Chemistry and Ames Laboratory, USDOE, Iowa State University, Ames, Iowa 50011 Steven A. Hofstadler,‡ Ljiljana Pasa-Tolic, and Richard D. Smith*
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Box 999, Richland, Washington 99352
On-line combination of capillary isoelectric focusing (CIEF) with electrospray ionization Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is demonstrated for high-resolution analysis of model proteins, human hemoglobin variants, and Escherichia coli proteins. The acquisition of high-resolution mass spectra of hemoglobin β chains allows direct identification of hemoglobin variants A and C, differing in molecular mass by 1 Da. Direct mass determination of cellular proteins separated in the CIEF capillary is achieved using their isotopic envelopes obtained from ESI-FTICR. The factors which dictate overall performance of CIEF-ESI-FTICR, including duty cycle, mass resolution, scan rate, and sensitivity, are discussed in the context of protein variants and cell lysates analyzed in this study. Significant advances in Fourier transform ion cyclotron resonance (FTICR) mass spectrometry over recent years involved an improved interface with external ion sources,1-4 novel ion manipulation techniques within the trapped ion cell,5-7 and enhanced data processing approaches.8,9 The advantages of FTICR include * To whom correspondence should be addressed. C.S.L.: phone (301) 4051020, fax (301) 405-1022, e-mail
[email protected]. R.D.S.: phone (509) 3760723, fax (509) 376-2303, e-mail: rd
[email protected]. † Present address: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742. ‡ Present address: Isis Pharmaceuticals, Inc., 2292 Faraday Ave., Carlsbad, CA 92008. (1) Lebrilla, C. B.; Amster, I. J.; McIver, R. T. Int. J. Mass Spectrom. Ion Processes 1989, 87, R7. (2) Kofel, P.; McMahon, T. B. Int. J. Mass Spectrom. Ion Processes 1990, 98, 1. (3) Beu, S. C.; Laude, D. A. Int. J. Mass Spectrom. Ion Processes 1991, 104, 109. (4) Hofstadler, S. A.; Schmidt, E.; Guan, Z.; Laude, D. A. J. Am. Soc. Mass Spectrom. 1993, 4, 168. (5) Gauthier, J. W.; Trautman, T. R.; Jacobson, D. B. Anal. Chim. Acta 1991, 246, 211. (6) Guan, S. H.; Kim, H. S.; Marshall, A. G.; Wahl, M. C.; Wood, T. D.; Xiang, X. Z. Chem. Rev. 1994, 94, 2161. (7) Bruce, J. E.; Anderson, G. A.; Smith, R. D. Anal. Chem. 1996, 68, 534. (8) Bruce, J. E.; Anderson, G. A.; Hofstadler, S. A.; Winger, B. E.; Smith, R. D. Rapid Commun. Mass Spectrom. 1993, 7, 700. (9) Guan, S. H.; Wahl, M. C.; Marshall, A. G. Anal. Chem. 1993, 65, 3647. S0003-2700(98)00224-8 CCC: $15.00 Published on Web 06/23/1998
© 1998 American Chemical Society
the ability to simultaneously realize ultrahigh MS resolution, mass measurement accuracy, and high sensitivity. Additionally, FTICR provides the capability for high-order tandem MS analyses for structural studies due to its nondestructive detection method.10-12 The combination of electrospray ionization (ESI) with FTICR was pioneered by McLafferty and co-workers.13 The use of ESIFTICR has demonstrated extensive potential for the characterization of biopolymers14 with part-per-million mass accuracy and mass resolving power exceeding 106. The ESI-FTICR results demonstrated the ability to distinguish species with similar masses and to identify adducts, posttranslational modifications, and substitutions. Resolution of the 1-Da spacing of peaks (primarily due to 13C isotopic distributions) allows unambiguous determination of charge (and thus mass) from a single charge state. The effective coupling of ESI with FTICR requires the transport of the ions between the ion source at atmospheric pressure and the trapped ion cell at a pressure below 10-9 Torr. The ESIFTICR instrumentation developed by Winger et al.15 allows rapid manipulation of pressures in the FTICR cell between those that appear optimum for ion trapping and cooling (i.e., >10-5 Torr) and those for high-resolution detection (105 L/s in close proximity to the trapped ion cell. Off-line combination of capillary zone electrophoresis (CZE) with FTICR using matrix-assisted laser desorption/ionization was demonstrated by Wilkins and co-workers.16 However, the mass (10) Marshall, A. G.; Grosshans, P. B. Anal. Chem. 1991, 63, A215. (11) Koster, C.; Kahr, M. S.; Castoro, J. A.; Wilkins, C. L. Mass Spectrom. Rev. 1992, 11, 495. (12) Buchanan, M. V.; Hettjch, R. L. Anal. Chem. 1993, 65, A245. (13) Henry, K. D.; Quinn, J. P.; McLafferty, F. W. J. Am. Chem. Soc. 1991, 113, 5447. (14) Beu, S. C.; Senko, M. W.; Quinn, J. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1993, 4, 190. (15) Winger, B. E.; Hofstadler, S. A.; Bruce, J. E.; Udseth, S. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 566.
Analytical Chemistry, Vol. 70, No. 15, August 1, 1998 3235
resolution achieved in that study was insufficient to resolve the 1-Da spacing of isotopic constituents within the singly and doubly charged species of somatostatin, equine myoglobin, and bovine insulin. In comparison with the ESI process, the multiple charging phenomenon inherent in ESI is particularly advantageous to the FTICR detection scheme, as resolving power is inversely proportional to m/z. Additionally, the use of the ESI process facilitates on-line interfacing of FTICR with separation methodologies including HPLC and CZE. Thus, the high pumping speed afforded by the cryopumping arrangement15 provides relatively rapid mass spectral acquisition rates and is crucial to on-line integration of ESI-FTICR with a high-speed separation technique such as CZE.17 Immediately after the first demonstration of on-line CZE-ESIFTICR,17 results have suggested that the combination may provide a nearly ideal approach for microsample analyses, owing to the inherent sensitivity of the technique and the enhanced information content available from high-resolution and high-precision mass measurements.18-20 High-resolution mass spectra (average resolution >45 000 full width at half-maximum, fwhm) of both the R and β chains of hemoglobin were acquired from the injection of 10 human erythrocytes, which corresponded to ∼4.5 fmol of hemoglobin.19 By employing sustained off-resonance irradiation5 for collisional dissociation of ions in the trapped ion cell, a partial amino acid sequence for the R chain of human hemoglobin was obtained from the injection of a population of 75 erythrocytes.20 High-order tandem MS measurements offered the potential for sensitive peptide fingerprinting and the ability to distinguish relatively small variations in biopolymer composition.18 In this study, on-line combination of capillary isoelectric focusing (CIEF) with ESI-FTICR is presented for the characterization of model proteins, human hemoglobin variants, and Escherichia coli proteins. The focusing effect of CIEF further permits analysis of dilute protein samples, with a typical 50-100fold concentration factor. With the full genome of several microorganisms having been sequenced and the sequencing of human genome well underway, capabilities for the analysis of the corresponding proteomes are attracting increased attention.21 Twodimensional analysis of cellular proteins using CIEF-ESI-FTICR provides a major step toward the comprehensive characterization of complex biological processes such as development, differentiation, and signal transduction in the field of cellular biochemistry. EXPERIMENTAL SECTION Materials and Chemicals. Model proteins, including cytochrome c (horse heart, pI 9.6), myoglobin (horse heart, pI 7.2 and 6.8), carbonic anhydrase I (human erythrocyte, pI 6.6), and carbonic anhydrase II (bovine erythrocyte, pI 5.9), were purchased from Sigma (St. Louis, MO). Another bovine carbonic anhydrase II sample obtained from Sigma exhibited a pI of 5.4. Human (16) Castoro, J. A.; Chiu, R. W.; Monnig, C. A.; Wilkins, C. L. J. Am. Chem. Soc. 1992, 114, 7571. (17) Hofstadler, S. A.; Wahl, J. H.; Bruce, J. E.; Smith, R. D. J. Am. Chem. Soc. 1993, 115, 6983. (18) Hofstadler, S. A.; Wahl, J. H.; Bakhtiar, R.; Anderson, G. A.; Bruce, J. E.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1994, 5, 894. (19) Hofstadler, S. A.; Swanek, F. D.; Gale, D. C.; Ewing, A. G.; Smith, R. D. Anal. Chem. 1995, 67, 1477. (20) Hofstadler, S. A.; Severs, J. C.; Smith, R. D.; Swanek, F. D.; Ewing, A. G. J. High Resolut. Chromatogr. 1996, 19, 617. (21) Kahn, P. Science 1995, 270, 369.
3236 Analytical Chemistry, Vol. 70, No. 15, August 1, 1998
hemoglobin variants A (pI 7.10), C (pI 7.50), S (pI 7.25), and F (pI 7.15) were acquired from Isolab (Akron, OH). The E. coli cells were suspended in a buffer which consisted of 10 mM Tris-HCl (pH 7.0), 5 mM magnesium chloride, 0.1 mM dithiothreitol, and 10% glycerol. The cells were disrupted by sonication for the release of cellular proteins.22 After sonication, DNase (Sigma) was added to a final concentration of 50 µg/mL for the digestion and removal of nucleic acids. The cellular proteins were collected in the supernatant following centrifugation at 2000g for 10 min. The protein solution was then desalted using a recently described microdialysis arrangement.23 The total protein concentration of the resulting solution, determined by the Bradford method (Bio-Rad kit, Richmond, CA), was approximately 12 mg/mL. Fused-silica capillaries with 50 µm i.d. and 192 µm o.d. (Polymicro Technologies, Phoenix, AZ) were coated internally with linear polyacrylamide for the elimination of electroosmotic flow and protein adsorption onto the capillary wall.24 Carrier ampholytes, Pharmalyte 3-10 and 5-8, were obtained from Pharmacia (Uppsala, Sweden). All chemicals, including acetic acid, ammonium acetate, ammonium hydroxide, dithiothreitol, glycerol, magnesium chloride, methanol, phosphoric acid, sodium hydroxide, and Tris-HCl, were acquired from Fisher (Fair Lawn, NJ). All background electrolytes and sample solutions were prepared using water purified by a Nanopure II system (Branstead, Dubuque, IA) and further filtered with a 0.22-µm membrane (Millipore, Bedford, MA). Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. The 7-T ESI-FTICR mass spectrometer utilized in this study was described in detail elsewhere.15 Briefly, ions were transferred from the ESI source, through a heated metal capillary, to the trapped ion cell by two sets of radio frequency (rf)-only quadrupoles. Background pressure in the trapped ion cell was maintained at 10-9-10-10 Torr by a custom cryopumping assembly consisting of two sets of cryobaffels with radiation shields which were maintained at 77 and 14 K, respectively, by closed-cycle cryogenic compressors. The large surface area of the cryobaffels provided pumping speeds in excess of 105 L/s, permitting rapid transitions between the high-efficiency, high-pressure ion accumulation (>10-5 Torr) and the low-pressure detection events (
