High-Sensitivity Electron Capture Dissociation Tandem FTICR Mass

Nick C. Polfer § , Kim F. Haselmann , Pat R. R. Langridge-Smith , Perdita E. Barran * ... Elisabet Carlsohn , Jonas Ångström , Mark R Emmett , Alan...
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Anal. Chem. 2001, 73, 3605-3610

High-Sensitivity Electron Capture Dissociation Tandem FTICR Mass Spectrometry of Microelectrosprayed Peptides Kristina Håkansson, Mark R. Emmett, Christopher L. Hendrickson, and Alan G. Marshall*,†

Center for Interdisciplinary Magnetic Resonance, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310

Electron capture dissociation (ECD) has previously been shown by other research groups to result in greater peptide sequence coverage than other ion dissociation techniques and to localize labile posttranslational modifications. Here, ECD has been achieved for 10-13-mer peptides microelectrosprayed from 10 nM (10 fmol/µL) solutions and for tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. Tandem Fourier transform ion cyclotron resonance (FTICR) mass spectra contain fragment ions corresponding to cleavages at all possible peptide backbone amine bonds, except on the N-terminal side of proline, for substance P and neurotensin. For luteinizing hormone-releasing hormone, all but two expected backbone amine bond cleavages are observed. The tandem FTICR mass spectra of the tryptic peptides contain fragment ions corresponding to cleavages at 6 of 12 (1545.7-Da peptide) and 8 of 21 (2944.5-Da peptide) expected backbone amine bonds. The present sensitivity is 200-2000 times higher than previously reported. These results show promise for ECD as a tool to produce sequence tags for identification of peptides in complex mixtures available only in limited amounts, as in proteomics. Tandem mass spectrometry (MS/MS)1 is a well-established technique for determining peptide primary sequences.2 In an MS/ MS experiment, a parent ion is first isolated from other ions of different mass-to-charge (m/z) ratios and then fragmented by (e.g.) high (keV)- or low (eV)-energy collision-induced dissociation (CID),3 infrared multiphoton dissociation (IRMPD),4 blackbody infrared radiative dissociation (BIRD),5 surface-induced dissociation (SID),6 or ultraviolet (UV) photodissociation.7,8 All of these † Member of the Department of Chemistry, Florida State University, Tallahassee, FL 32310. (1) McLafferty, F. W. In Tandem Mass Spectrometry; McLafferty, F. W., Ed.; Wiley: New York, 1983. (2) Biemann, K.; Martin, S. A. Mass Spectrom. Rev. 1987, 6, 1-76. (3) Hayes, R. N.; Gross, M. L. Methods Enzymol. 1990, 193, 237-263. (4) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (5) Price, W. D.; Schnier, P. D.; Williams, E. R. Anal. Chem. 1996, 68, 859866. (6) Tsaprailis, G.; Nair, H.; Somogyi, A.; Wysocki, V. H.; Zhong, W.; Futrell, J. H.; Summerfield, S. G.; Gaskell, S. J. J. Am. Chem. Soc. 1999, 121, 51425154.

10.1021/ac010141z CCC: $20.00 Published on Web 07/06/2001

© 2001 American Chemical Society

techniques result mainly in cleavage of the peptide amide bonds, providing N-terminal b-type and C-terminal y-type ions.9 The peptide sequence can then be read from the mass differences between the fragment ions. The highest sensitivity peptide tandem mass spectrometry has been achieved by use of low-energy CID. Andren et al. demonstrated a low-attomole detection limit for a solution-phase peptide by use of microelectrospray ionization10 with a triple quadrupole mass spectrometer,11 and Morris et al. achieved a high-attomole detection limit with a quadrupole/time-of-flight instrument with on-line electrospray ionization liquid chromatography.12 Valaskovic et al. achieved similar sensitivity for a large protein by coupling capillary electrophoresis, nanoelectrospray ionization,13,14 and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry15 and fragmenting the protein by collisions in the ion source.16 However, the latter approach did not allow for isolation of a precursor ion; ergo, a pure sample was required. Wilm et al. also demonstrated sequencing of femtomoles of proteins from polyacrylamide gels by CID tandem mass spectrometry with a triple quadrupole instrument.17 However, the major drawback of low-energy CID is that only limited energy is available to activate the parent ions.3 Cleavage thus tends to occur primarily at the weakest bonds: in peptides, on the N-terminal side of a proline residue18 or on the C-terminal side of an aspartic acid.19 As a (7) Bowers, W. D.; Delbert, S.-S.; Hunter, R. L.; McIver, R. T., Jr. J. Am. Chem. Soc. 1984, 106, 7288-7289. (8) Williams, E. R.; Furlong, J. J. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1990, 1, 288-294. (9) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601-601. (10) Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 605613. (11) Andren, P. E.; Emmett, M. R.; Caprioli, R. M. J. Am. Soc. Mass Spectrom. 1994, 5, 867-869. (12) Morris, H. R.; Paxton, T.; Dell, A.; Langhorne, J.; Berg, M.; Bordoli, R. S.; Hoyes, J.; Bateman, R. H. Rapid Commun. Mass Spectrom. 1996, 10, 889896. (13) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (14) Wilm, M. S.; Matthias, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167-180. (15) Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. Mass Spectrom. Rev. 1998, 17, 1-35. (16) Valaskovic, G. A.; Kelleher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (17) Wilm, M.; Shevchenko, A.; Houthaeve, T.; Breit, S.; Schweigerer, L.; Fotsis, T.; Mann, M. Nature 1996, 379, 466-469. (18) Vekey, K. Mass Spectrom. Rev. 1995, 14, 195-225. (19) Yu, W.; Vath, J. E.; Huberty, M. C.; Martin, S. A. Anal. Chem. 1993, 65, 3015-3023.

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consequence, it is rare to obtain a complete peptide sequence from its CID mass spectrum. Electron capture dissociation (ECD)20,21 is a relatively new technique that offers more extensive and nonspecific fragmentation, resulting in greater (and in many cases complete) peptide sequence coverage.22,23 ECD cleaves peptide backbone amine bonds, to produce N-terminal c-type and C-terminal z•-type ions,9 in contrast to the b and y ions typically produced by other techniques. ECD is therefore a valuable complementary fragmentation technique. The only exceptions from the nonspecificity of ECD are the following: bonds on the N-terminal side of a proline residue are not cleaved, and there is a preference for cleavage on the C-terminal side of tryptophan.24 ECD is also unique in its ability to cleave disulfide bonds.25 Moreover, ECD can cleave peptide backbone amine bonds of posttranslationally modified peptides with limited effect on modifications, allowing for unambiguous location of modifications (e.g., glycosylation, phosphorylation).26-28 Finally, ECD preceded by collisional activation, so-called activated ion ECD,29 improves the extent of sequence coverage in tandem MS of large proteins. ECD is performed by irradiating multiply charged positive ions, produced by electrospray,30,31 with low-energy electrons.21 The electrons and ions must move with very low relative velocities for long enough to interact. Those requirements are fulfilled in the FTICR mass analyzer, in which ions may be trapped for extended time periods.15 FTICR mass spectrometry adds the advantages of ultrahigh resolving power32,33 and mass accuracy.34-36 Recent work has also shown that FTICR mass spectrometry reaches a detection limit comparable to those of other mass analyzers.37-39 However, in its simplest experimental configuration, (20) Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Anal. Chem. 2000, 72, 563-573. (21) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (22) Kruger, N. A.; Zubarev, R. A.; Horn, D. M.; McLafferty, F. W. Int. J. Mass Spectrom. 1999, 185/186/187, 787-793. (23) Axelsson, J.; Palmblad, M.; Hakansson, K.; Hakansson, P. Rapid Commun. Mass Spectrom. 1999, 13, 474-477. (24) Kruger, N. A.; Zubarev, R. A.; Carpenter, B. K.; Kelleher, N. L.; Horn, D. M.; McLafferty, F. W. Int. J. Mass Spectrom. 1999, 182/183, 1-5. (25) Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W. J. Am. Chem. Soc. 1999, 121, 28572862. (26) Kelleher, N. L.; Zubarev, R. A.; Bush, K.; Furie, B.; Furie, B. C.; McLafferty, F. W.; Walsh, C. T. Anal. Chem. 1999, 71, 4250-4253. (27) Mirgorodskaya, E.; Roepstorff, P.; Zubarev, R. A. Anal. Chem. 1999, 71, 4431-4436. (28) Stensballe, A.; Norregaard-Jensen, O.; Olsen, J. V.; Haselmann, K. F.; Zubarev, R. A. Rapid Commun. Mass Spectrom. 2000, 14, 1793-1800. (29) Horn, D. M.; Ge, Y.; McLafferty, F. W. Anal. Chem. 2000, 72, 4778-4784. (30) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F. Mass Spectrom. Rev. 1990, 9, 37-70. (31) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Science 1989, 246, 64-71. (32) Kelleher, N. L.; Senko, M. W.; Siegel, M. M.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1997, 8, 380-383. (33) Shi, S. D.-H.; Hendrickson, C. L.; Marshall, A. G. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 11532-11537. (34) Green, M. K.; Vestling, M. M.; Johnston, M. V.; Larsen, B. S. Anal. Biochem. 1998, 260, 204-211. (35) Bruce, J. E.; Anderson, G. A.; Wen, J.; Harkewicz, R.; Smith, R. D. Anal. Chem. 1999, 71, 2595-2599. (36) He, F.; Hendrickson, C. L.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2000, 11, 120-126. (37) Belov, M. E.; Gorshkov, M. V.; Udseth, H. R.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2271-2279.

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the efficiency of ECD to convert parent ions into product ions is lower than that of CID, “a problem for very limited sample sizes”.22 Here, we show that ECD fragment spectra containing complete or nearly complete sequence information can be successfully achieved from 10 nM (10 fmol/µL) concentrations of 10-13-mer peptides in a nonoptimized experimental geometry. Also, we demonstrate partial sequence coverage for two tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein. That sensitivity, namely, 200-2000 times higher than previously achieved,27,29 opens up new possibilities for obtaining sequence tags to identify proteins in complex mixtures, as for example in proteomics.40-44 EXPERIMENTAL SECTION Sample Preparation. The bioactive peptides, substance P, human luteinizing hormone-releasing hormone (LHRH), and neurotensin were purchased from Sigma (St. Louis, MO). Stock solutions of 1000 pmol/µL (1 mM) were prepared in HPLC grade water (J. T. Baker, Philipsburg, NJ). Dilution of the stock solutions was performed in steps of 10 down to a concentration of 10 fmol/ µL (10 nM). In all steps but the last, 10 µL of the peptide solution was placed in 90 µL of water. For the last step, the solvent was 50:50 methanol (Baker) and water with 0.25% acetic acid (Aldrich, Milwaukee, WI). A 28-kDa protein digest was prepared by dissolving 10 nmol of the lectin from Erythrina corallodendron (Sigma) in 500 µL of buffer containing 0.1 mM CaCl2 and 0.1 M NH4HCO3, to which 100 pmol of modified trypsin (Promega, Madison, WI) had been added. Digestion of the protein proceeded at 38 °C for 4 h. The mixture was dried in a Speedvac (Savant, Inc., Farmingdale, NY), and the dried residue was dissolved in 100 µL of water to yield a concentration of 100 pmol/µL. This stock solution was then diluted as described above to a final concentration of 50 fmol/µL in 50:50 methanol/water with 0.25% acetic acid. All solutions were kept on ice until run in the mass spectrometer. FTICR Mass Spectrometry. FTICR mass spectrometry was performed with a home-built instrument, constructed around a 7-T, unshielded superconducting magnet (Oxford Instruments, Oxford, U.K.).45 The peptide solutions were infused by use of a syringe pump at a flow rate of 300 nL/min and ionized by microelectrospray.10 The electrospray emitter consisted of a 50µm-i.d. fused-silica capillary which had been mechanically ground to a uniform thin-walled tip.46 In the present version of the 7-T instrument, ions are transported into the mass spectrometer (38) Martin, S. E.; Shabanowitz, J.; Hunt, D. F.; Marto, J. A. Anal. Chem. 2000, 72, 4266-4274. (39) Quenzer, T. L.; Emmett, M. R.; Hendrickson, C. L.; Kelly, P. H.; Marshall, A. G. Anal. Chem. 2001, 73, 1721-1725. (40) Borman, S. Chem. Eng. News 2000, 78 (31), 31-37. (41) Shevchenko, A.; Jensen, O. N.; Podtelejnikov, A. V.; Sagliocco, F.; Wilm, M.; Vorm, O.; Mortensen, P.; Shevchenko, A.; Boucherie, H.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445. (42) Jensen, O. N.; Wilm, M.; Shevchenko, A.; Mann, M. Methods Mol. Biol. 1999, 112, 571-588. (43) Yates, J. R. I. Trends Genet. 2000, 16, 5-8. (44) Gygi, S. P.; Corthals, G. L.; Zhang, Y.; Rochon, Y.; Aebersold, R. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9890-9895. (45) White, F. M.; Marto, J. A.; Marshall, A. G. Rapid Commun. Mass Spectrom. 1996, 10, 1845-1849. (46) Quinn, J. P.; Emmett, M. R.; Marshall, A. G. 46th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, 1998; pp 1388-1388

Figure 1. Schematic diagram of the apparatus for high-sensitivity electron capture dissociation experiments. A heated-filament electron gun is placed ∼1 m behind the ICR cell in a 7-T Fourier transform ion cyclotron resonance mass spectrometer.

through a Chait-style atmosphere-to-vacuum interface47 and externally accumulated48 in a short (15-cm) octapole. After accumulation, the collected ions are transferred through a hexapole ion guide and captured by gated trapping in an open cell49 with rectangular electrodes. For high-sensitivity ECD, ions were accumulated for 6-8 s to generate a sufficient number of parent ions for the fragmentation experiments. Peptide ion dissociation or proton stripping, which can occur if large ion populations are stored inside the external multipole,50,51 was not observed during the accumulation event. Following chirp excitation, the trapped ions were detected by direct-mode broadband detection to yield 256 (pure peptides) or 512 Kword (tryptic digest) time-domain data points. The excitation parameters were 72-540 kHz at 350 Hz/µs, 60 Vp-p amplitude for pure peptides and 43-1100 kHz at 350 Hz/µs, 80 Vp-p amplitude for the digest. All experimental transients were subjected to Hanning apodization and one zero fill prior to fast Fourier transform (FFT) followed by magnitude calculation. The spectra were externally calibrated52,53 by use of an electrospray calibration mixture (Agilent Technologies, Wilmington, DE). The entire experimental event sequence was controlled by an Odyssey data station (FinniganThermoQuest Corp., Bremen, Germany). Electron Capture Dissociation. A heated-filament electron gun was installed ∼1 m behind the ICR cell (see Figure 1). The filament is biased at +3.1 V applied to one end of the filament and the heating current adjusted to ∼3.8 A to produce a voltage drop of 4.3 V across the filament. Reproducible performance requires keeping the measured voltage drop across the filament the same from day to day, rather than keeping the heating current constant. The same heating current does not necessarily result in exactly the same voltage drop every time, due to fluctuations (47) Chowdhury, S. K.; Katta, V.; Chait, B. T. Rapid Commun. Mass Spectrom. 1990, 4, 81-87. (48) Senko, M. W.; Hendrickson, C. L.; Emmett, M. R.; Shi, S. D.-H.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1997, 8, 970-976. (49) Beu, S. C.; Laude, D. A., Jr. Int. J. Mass Spectrom. Ion Processes 1992, 112, 215-230. (50) Sannes-Lowery, K. A.; Griffey, R. H.; Kruppa, G. H.; Speir, J. P.; Hofstadler, S. A. Rapid Commun. Mass Spectrom. 1998, 12, 1957-1961. (51) Hakansson, K.; Axelsson, J.; Palmblad, M.; Hakansson, P. J. Am. Soc. Mass Spectrom. 2000, 11, 210-217. (52) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 27442748. (53) Shi, S. D.-H.; Drader, J. J.; Freitas, M. A.; Hendrickson, C. L.; Marshall, A. G. Int. J. Mass Spectrom. 2000, 195/196, 591-598.

in temperature that affect the filament resistance. A possible explanation for the importance of a constant voltage drop might be related to the strong increase in electron capture cross section with decreasing electron energy: energy below 0.2 eV is desirable.21 In the present experimental configuration, the electron energy is determined by the potential difference between the filament and the rear trapping electrodes of the ICR cell. In ECD experiments, the trapping electrodes are kept at +1.5 V and the filament is a voltage divider with +3.1 V at one end and -1.2 V at the other. Electrons with the right energy to be captured by positively charged ions inside the cell likely originate from a small segment of the filament. If the voltage drop changes, the filament location at which electrons with the right energy are produced will change. Another parameter critical to the success of the ECD experiment is the voltage applied to a collector plate located behind the filament (Figure 1). That plate is normally grounded and used to measure filament emission current. However, no such measurements are performed in the present experiments. If the collector plate is left grounded, no ECD is observed, but application of a small negative voltage (-2 V) to the collector achieves successful and reproducible ECD fragmentation. A possible explanation is that electrons are emitted in all directions from the filament, so that only a fraction of them will propagate all the way to the ICR cell. However, with mild initial acceleration toward the cell, the number of electrons that can interact with the trapped ions may increase. We attempted to monitor the electron current through the cell at the optimum ECD parameters by measuring the current on the transfer hexapole, located at the opposite side of the cell with respect to the filament. However, the current was too small to register with a standard picoammeter. All ECD experiments were performed without pulsing gas into the system and without extra electrodes for trapping the electrons inside the cell. To perform ECD, we first isolated a parent ion by storedwaveform inverse Fourier transform (SWIFT) ejection54,55 and then irradiated with electrons for 20-30 s, followed by chirp excitation and broadband detection of the fragment ions. The doubly protonated precursor ions were chosen for substance P and LHRH, whereas the triply protonated ion was used for neurotensin. From the tryptic digest, a doubly protonated peptide of m/z 773.9, corresponding to the amino acids 37-50 in the lectin, and a triply protonated peptide of m/z 982.5, corresponding to the amino acids 74-99, were chosen for ECD. All presented spectra were obtained by averaging 30 time-domain transients over ∼15-20 min, consuming ∼45-60 fmol of the pure peptides and ∼300 fmol of each tryptic peptide. Between irradiation events, a grid located between the filament and the cell is kept at -20 V, preventing electrons from entering the cell. During irradiation, the grid voltage is +20 V (see Figure 1). RESULTS AND DISCUSSION Substance P. The ECD spectrum obtained from the doubly protonated ion of substance P, electrosprayed from a 10 fmol/µL (i.e., 10 nM) solution of the peptide, is shown in Figure 2. The figure also displays the peptide sequence, including its C-terminal (54) Marshall, A. G.; Wang, T.-C. L.; Ricca, T. L. J. Am. Chem. Soc. 1985, 107, 7893-7897. (55) Guan, S.; Marshall, A. G. Int. J. Mass Spectrom. Ion Processes 1996, 157/ 158, 5-37.

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Figure 2. Electron capture dissociation fragment FTICR mass spectrum obtained from a 10 fmol/µL sample of microelectrosprayed substance P (8-s accumulation, 30 scans). The doubly protonated ion is SWIFT isolated and irradiated with electrons for 30 s. The observed fragment ions correspond to cleavages at all peptide backbone amine bonds except for the N-terminal side of proline.

Figure 3. Electron capture dissociation fragment FTICR mass spectrum obtained from a 10 fmol/µL sample of microelectrosprayed LHRH (8-s accumulation, 30 scans). The doubly protonated LHRH is SWIFT isolated and irradiated with electrons for 30 s. All expected backbone amine bond cleavages are observed, except the two closest to the N-terminus. The pE at the N-terminus of the peptide denotes pyroglutamic acid.

amidation. Seven c ions and one z• ion are observed, corresponding to cleavages at all peptide backbone amine bonds, except those on the N-terminal side of proline. The latter cleavages are not expected because proline has a cyclic structure, so that the peptide backbone would still be connected through the side chain even if the backbone amine bond is cleaved. The predominance of N-terminal c ions can be understood from the substance P sequence. The N-terminal amino acid residue is arginine, the most basic amino acid, and therefore most likely to carry the charge. As a consequence, the complementary z• products are neutral and cannot be detected as ions in the mass spectrometer. The observation of the z9• ion can also be explained because, in addition to arginine, there is another basic amino acid residue (lysine) at position three in the peptide sequence. Localization of the charge on the lysine residue and cleavage of the backbone amine bond between the first proline and the lysine creates the z9• ion. In addition to the c and z• fragment ions, another singly charged ion is observed at m/z 1348.7, namely, 1 Da higher than a singly protonated substance P ion and corresponding instead to a doubly protonated molecular ion that has captured an electron, to give a total charge of 1 without fragmentation. LHRH. Figure 3 shows the ECD spectrum obtained from the doubly protonated ion of LHRH, electrosprayed from a 10 nM solution of the peptide. This peptide has an N-terminal pyroglutamic acid (pE) and a C-terminal amidation. In contrast to the substance P ECD mass spectrum, mostly C-terminal ions are observed. Again, this behavior can be understood from the peptide 3608

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sequence. The third amino acid residue, counting from the C-terminus, is an arginine, the most likely protonated site. As a consequence, all N-terminal products, except c9, will be neutral (c8 is not observed because it corresponds to cleavage on the N-side of proline). Accordingly, c9 is the only c ion in the ECD mass spectrum. The observed fragment ions correspond to cleavage of all expected peptide backbone amine bonds, except for the two closest to the N-terminus. LHRH contains a tryptophan residue, which in other experiments has been shown to be a favored site for cleavage.24 However, for LHRH, there is no preference for cleavage at this site, which yields the observed z7• ion. A possible explanation is that LHRH has a compact, folded structure in the gas phase, so that accessibility of the tryptophan carbonyl oxygen is sterically hindered.56 The carbonyl oxygens are involved in the proposed ECD cleavage mechanism,25 in which, after electron capture, an energetic hydrogen atom is released and subsequently captured by a site of high hydrogen atom affinity, such as a carbonyl oxygen. Support for a highly folded gas-phase LHRH structure has been obtained by hydrogen/deuterium exchange experiments, in which LHRH did not display any observable exchange.57 Also, the collisional cross section of doubly protonated LHRH has been measured to be smaller than expected from molecular dynamics calculations.58 Another unexpected behavior is observed for the remaining z• ions. Figure 4 (top) shows mass-scale expansion for the z6• region. The mass of the monoisotopic peak does correspond to the expected monoisotopic mass of the z6• radical cation. However, the magnitude of the nuclide 1 Da higher is too high to correspond to the substitution of one 13C or 15N atom. Thus, a mixture of two species whose monoisotopic masses differ by 1 Da must be present. Figure 4 (middle) shows a mass scale expansion for the z3• region. This time, the magnitude of the monoisotopic z3• radical ion peak is much lower than that of the peak 1 Da higher in mass, designated as z3. A 1-Da mass increase may be explained by the capture of a hydrogen atom to form a more stable even-electron species from the radical z• ion. Even-electron z ions have been observed previously.20,22,27 However, in those experiments, either the yield of the even-electron species was reported to be small20,22 or a mass decrease of 1 Da was observed.27 At present, there is not a complete consensus regarding the mechanism behind the proposed hydrogen atom capture. An alternative explanation proposed by Zubarev et al.20 for the observed even-electron z ions is that the released hydrogen atom is captured by the backbone R-carbon, followed by the formation of a z/c• fragment pair. Figure 4 (bottom) shows an even more extreme example. The expected monoisotopic z5• radical cation is not observed at all. Instead, a species 1 Da heavier, designated as z5, is seen. The same is true for the z4• ion (not shown). This behavior is also indicated in Figure 3, in which the ions labeled as z4 and z5 are observed at masses 1 Da higher than those of the z4• and z5• radical ions. Neurotensin. The ECD FTICR mass spectrum of a 10 fmol/ µL solution of neurotensin, a 13-residue peptide containing an (56) Breuker, K.; Horn, D. M.; Cerda, B.; McLafferty, F. W. 48th ASMS Conference on Mass Spectrometry and Allied Topics Long Beach, CA, 2000; poster presentation. (57) Freitas, M. A.; Hendrickson, C. L.; Emmett, M. R.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 1998, 9, 1012-1019. (58) Gill, A. C.; Jennings, K. R.; Wyttenbach, T.; Bowers, M. T. Int. J. Mass Spectrom. 2000, 195/196, 685-697.

Figure 4. Mass scale-expanded segments for each of three C-terminal fragment ions from an FTICR mass spectrum following electron capture dissociation of microelectrosprayed LHRH. For z6• (top) and z3• (middle), a mixture of two species differing by one mass unit is observed; for z5•, the mass shifts by 1 Da (bottom), possibly due to capture of a hydrogen atom to form an even-electron z ion from the radical z• ion (see text).

Figure 5. Electron capture dissociation fragment FTICR mass spectrum obtained from a 10 fmol/µL sample of microelectrosprayed neurotensin (6-s accumulation, 30 scans). The triply protonated neurotensin is SWIFT isolated and irradiated with electrons for 20 s. All expected backbone amine bond cleavages are observed. The pE at the N-terminus of the peptide denotes pyroglutamic acid.

N-terminal pyroglutamic acid, is shown in Figure 5. ECD of the triply protonated precursor ion results in both singly and doubly charged fragment ions. Six c and seven z• ions are observed. Another major product is the charge-reduced species [M + 3H + e-]2+. The observed fragment ions correspond to cleavage at all of the expected peptide backbone amine bonds. The z8•, z92+•, and z102+• ions all behave similarly to the z• ions from LHRH; namely, a shift in the isotopic distribution, indicating hydrogen atom capture to form even-electron species is observed. Tryptic Peptides from an Unfractionated Digest of a 28kDa Protein. Figure 6 (bottom) shows the ESI FTICR mass spectrum for a 50 nM unfractionated tryptic digest of the lectin from E. corallodendron. Nine peaks could be matched to theoretical masses of lectin tryptic peptides. ECD spectra of the doubly protonated peptide of m/z 773.9 and of the triply protonated peptide of m/z 982.5 are shown at the top of the figure. The m/z

Figure 6. FTICR mass spectrum of a 50 nM tryptic digest of the lectin from E. corallodendron (bottom). Tandem electron capture dissociation of the doubly protonated peptide at m/z 773.9 (top left) and the triply protonated peptide at m/z 982.5 (top right) show partial sequence coverage (50% and 38% backbone amine bond cleavage, respectively). The peaks labeled 2 and 3 in the ECD spectrum of the m/z 773.9 peptide denote second and third harmonics of the parent ion.

773.9 peptide corresponds to the amino acids 37-50 in the lectin and has the sequence,

The ECD cleavage sites are indicated. Six of 12 expected backbone amine bonds are cleaved, resulting in a sequence tag of 5 amino acids. All of the observed ECD fragments are C-terminal z• ions, explained as for LHRH: the C-terminal arginine residue carries the remaining proton. As for LHRH, the sequence coverage of the m/z 773.9 tryptic peptide is not complete. It has previously been pointed out that the abundance of radical z• ions is typically about half that of more stable even-electron c ions.27 The lower abundance of z• ions poses a challenge to the use of ECD for sequencing tryptic peptides at low concentrations. Trypsin cleaves a protein backbone on the C-terminal side of a basic amino acid residue (lysine or arginine), resulting in peptides with a basic residue at their C-termini. Such peptides are most likely to form z• ions after electron capture. Another similarity between the m/z 773.9 tryptic peptide and LHRH is that they both contain a tryptophan residue. Surprisingly, ECD fragments corresponding to cleavage on the C-terminal side of tryptophan are not observed at all. The m/z 982.2 tryptic fragment corresponds to the amino acids 74-99 in the lectin and has the sequence,

Here, 8 of 21 expected backbone amine bonds are cleaved. As for neurotensin, whose parent ion is also triply protonated, Analytical Chemistry, Vol. 73, No. 15, August 1, 2001

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both c and z• ions are observed. The two lowest-mass z ions (z4 and z5) are even-electron species. The limited sequence coverage can be attributed to the size of the peptide (2944.5 Da). For such a large peptide, the number of ECD fragmentation pathways is larger and the low percentage of parent ions converted to products in the current experimental configuration will spread over all of those channels. CONCLUSION Tandem mass spectra containing fragment ions corresponding to cleavages of all possible peptide backbone amine bonds, except for cleavage at the N-terminal side of proline, have been obtained by electron capture dissociation of substance P and neurotensin ions generated from 10 nM (10 fmol/µL) samples. For LHRH, all expected backbone amine bond cleavages, except two, are observed at the same conditions. ECD spectra of two tryptic peptides from a 50 nM unfractionated digest of a 28-kDa protein yield partial sequence coverage (50% and 38% of the backbone amine bonds cleaved, respectively). The presently achieved ECD sensitivity is 200-2000 times higher than previously reported. This advance opens up new

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possibilities for applying ECD to problems in which the amount of sample is limited or the peptide is present at low concentration. Two obvious examples are proteomics and the analysis of peptides at endogenous levels in biological fluids.

ACKNOWLEDGMENT We thank John P. Quinn for his assistance and valuable suggestions. We also thank Carol L. Nilsson for providing the lectin digest. We acknowledge Guillaume van der Rest, Terri L. Quenzer, and Fei He for helpful discussions. This work was supported by the NSF National High Field FT-ICR Facility (CHE99-09502), Florida State University, the National High Magnetic Field Laboratory, and the Swedish Foundation for International Cooperation in Research and Higher Education (STINT).

Received for review January 31, 2001. Accepted June 1, 2001. AC010141Z