Ion Activation in Electron Capture Dissociation To Distinguish between

Sep 18, 2007 - Biomolecular Mass Spectrometry Laboratory, Swiss Federal Institute of Technology in Lausanne, Lausanne, CH-1015, Switzerland, Ion Cyclo...
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Anal. Chem. 2007, 79, 7596-7602

Ion Activation in Electron Capture Dissociation To Distinguish between N-Terminal and C-Terminal Product Ions Yury O. Tsybin,*,† Huan He,‡,§ Mark R. Emmett,‡,§ Christopher L. Hendrickson,‡,§ and Alan G. Marshall‡,§

Biomolecular Mass Spectrometry Laboratory, Swiss Federal Institute of Technology in Lausanne, Lausanne, CH-1015, Switzerland, Ion Cyclotron Resonance Program, National High Magnetic Field Laboratory, Florida State University, 1800 East Paul Dirac Drive, Tallahassee, Florida 32310-4005, and Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida 32306

We present a method to distinguish N-terminal from C-terminal product ions in electron capture dissociation (ECD) MS/MS due to the change in relative abundances of even-electron (prime) and odd-electron (radical) product ions produced in consecutive ECD and activated ionECD mass spectra. The method is based on the rate and direction of hydrogen atom transfer between N-terminal and C-terminal ECD products and its dependence on ion internal energy. We demonstrate that increasing ion internal energy by vibrational activation prior to ECD results in decreased ratio of radical/prime N-terminal product ions (c•/c′ ratio), but increased ratio of radical/ prime C-terminal product ions (z•/z′ ratio) in many cases. The combination of AI-ECD and ECD promises to increase the confidence of mass spectrometry-based peptide sequencing and protein identification.

Modern proteomics technologies based on tandem mass spectrometry (MS/MS) typically depend on identification of either b (N-terminal) and y (C-terminal) ions (resulting from cleavage of the carbonyl carbon-nitrogen backbone bond, produced by collision-induced dissociation (CID)1 or infrared multiphoton dissociation (IRMPD)2), or c (N-terminal) and z (C-terminal) ions (formed by cleavage of the N-Ca backbone bond, produced by electron capture dissociation (ECD)3 or electron-transfer dissociation (ETD)4). However, because one does not know in advance whether a given MS/MS product ion is N-terminal or C-terminal, peptide identification can be ambiguous. Considerable improvement in the confidence of protein identification becomes possible if N-terminal (and/or C-terminal) * To whom correspondence should be addressed. E-mail: [email protected]. † Swiss Federal Institute of Technology in Lausanne. ‡ National High Magnetic Field Laboratory, Florida State University. § Department of Chemistry and Biochemistry, Florida State University. (1) Wells, J. M.; McLuckey, S. A. Biol. Mass Spectrom. 2005, 402, 148-185. (2) Little, D. P.; Speir, J. P.; Senko, M. W.; O’Connor, P. B.; McLafferty, F. W. Anal. Chem. 1994, 66, 2809-2815. (3) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265-3266. (4) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528-9533.

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fragment ions resulting from cleavage between the same pair of residues (so-called “golden pair” fragment ions) can be observed in both CID (IRMPD) and ECD (ETD).5 Because y-ions are typically 16.019 Da higher in mass than z•-ions cleaved between the same pair of residues, and the corresponding c′-ions are 17.027 Da higher in mass than b-ions, observation of either of those mass differences allows for confident type assignment of N-terminal (b and c′) or C-terminal (y and z•) fragment ions in protein analysis.5,6 Knowledge of the precursor ion mass (obtained from equations b + y ) M or c′ + z• ) M) further increases the reliability of product ion type assignment.6 The prime (e.g., c′) and radical (e.g., z•) notation distinguishes fragment ions differing by one H-atom.6 However, in top-down mass spectrometry of multiply charged proteins, CID (IRMPD) typically cleaves at far fewer peptide linkages than ECD,7 and in bottom-up mass spectrometry, ECD and CID product ions do not commonly occur with similar yield at the same cleavage site in numerous tryptic peptides.8 The presence of abundant radical c-type ions in peptide analysis further complicates correct product ion type assignment and additional computational efforts are required.9 Nevertheless, application of the golden pair rules to MS/MS of peptides produced a substantial improvement for the combined ECD/CID versus separate CID or ECD approach for identification of proteins from a protein digest (Escherichia coli lysate) by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS).10 To further improve the automatic integration of ECD mass spectral data into database search engines, an extensive study of 15 000 ECD MS/MS data from tryptic peptide dications revealed several rules for hydrogen atom abstraction to and from z• ions.9 (5) Horn, D. M.; Zubarev, R. A.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 10313-10317. (6) Zubarev, R. Expert Rev. Proteomics 2006, 3, 251-261. (7) Zubarev, R. A.; Fridriksson, E. K.; Einar, K.; Horn, D. M.; Kelleher, N. L.; Kruger, N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. Mass spectrometry in biology and medicine; Humana Press: Totowa, NJ, 2000. (8) Savitski, M. M.; Kjeldsen, F.; Nielsen, M. L.; Zubarev, R. A. Angew. Chem., Int. Ed. 2006, 45, 5301-5303. (9) Savitski, M. M.; Kjeldsen, F.; Nielsen, M. L.; Zubarev, R. A. J. Am. Soc. Mass Spectrom. 2007, 18, 113-120. (10) Nielsen, M. L.; Savitski, M. M.; Zubarev, R. A. Mol. Cell. Proteomics 2005, 4, 835-845. 10.1021/ac071165u CCC: $37.00

© 2007 American Chemical Society Published on Web 09/18/2007

Figure 1. Pathways for separation of complementary products from [c′ + z•] complex in ECD/ETD as a function of complex ion lifetime. Ion internal energy of both precursor and radical intermediate ions can be varied by vibrational activation with IR laser irradiation or by energetic collisions.

MS/MS specificity and efficiency depend not only on the dissociation technique (CID, IRMPD, and ECD) but also on the rate of energy deposition into the precursor ion. For example, in ECD of multiply charged precursor cations, formation of a charged radical complex of ECD products ([c′ + z•]complex) is believed to be the initial result of an ion-electron interaction, Figure 1.9,11,12 The subsequent separation of charged or neutral products from the [c′ + z•]complex will proceed if enough internal energy is available.9 The strengths of the bonds between the products determine the lifetime of the complex. Peptide and protein gasphase conformation in general and amino acid side-chain interactions (hydrophobic-hydrophobic, hydrophilic-hydrophilic, hydrophobic-hydrophilic, ionic-aromatic interactions, etc.) and side-chain-backbone interactions in particular influence the binding force between ECD products to be separated and therefore the lifetime of the [c′ + z•] complex. The lifetimes of the radical intermediate complexes can be estimated by, for example, a double resonance ECD method, as shown by O’Conner and coworkers.11 The extent of hydrogen atom abstraction monitored in hydrogen-deuterium exchange experiments provides further correlation with the lifetimes of radical intermediate complexes.13 Influence of ion internal energy (IR laser and blackbody irradiation, collisions with neutral molecules, and energetic electron impact) on total ECD product ion relative abundance has been exploited in peptide and protein structural analysis.14,15 Modulation of product ion yield in activated ion-ECD (AI-ECD) as a function (11) Lin, C.; Cournoyer, J. C.; O’Connor, P. B. J. Am. Soc. Mass Spectrom. 2006, 17, 1605-1615. (12) Breuker, K.; Oh, H. B.; Lin, C.; Carpenter, B. K.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 14011-14016. (13) O’Connor, P. B.; Lin, C.; Cournoyer, J. J.; Pittman, J. L.; Belyayev, M.; Budnik, B. A. J. Am. Soc. Mass Spectrom. 2006, 17, 576-585. (14) Breuker, K.; Oh, H. B.; Horn, D. M.; Cerda, B. A.; McLafferty, F. W. J. Am. Chem. Soc. 2002, 124, 6407-6420. (15) Horn, D. M.; Breuker, K.; Frank, A. J.; McLafferty, F. W. J. Am. Chem. Soc. 2001, 123, 9792-9799.

of ion internal energy provides more extensive sequence coverage for analysis of primary, secondary, and tertiary structure of gas-phase proteins,14,16 including disulfide bond connectivity.17,18 The free-radical cascade mechanism strongly suggests that secondary fragmentation in ECD is a function of ion internal energy.19 Despite AI-ECD applications based on total product ion abundance mentioned above, the influence of ion activation on hydrogen atom transfer between ECD products leading to variation of radical and prime components has not been substantially explored. Here, we show that AI-ECD increases the abundance of c′-ions relative to c•-ions and decreases the abundance of z′ relative to z•-ions, compared to ECD. Understanding this effect requires systematic study of the radical/prime ECD product ion ratio as a function of ion internal energy. Preliminary prior results include dependence of radical/prime product ion ratio in ECD conducted under different electron injection conditions (multiple vs single pass mode).20,21 Here, we demonstrate the important role of ion activation prior to ECD (Figure 1) on hydrogen atom transfer between ECD products and subsequent variation in radical/prime product ion ratio. We also present support for the above-described ECD product ion formation model, Figure 1. Combined AI-ECD and ECD MS/MS augurs for improved peptide and protein analysis, based on automated product ion type determination for improved de novo peptide sequencing in proteomics. METHODS Sample Preparation. Standard peptides and proteins as well as synthetic peptides were obtained from Sigma Aldrich, including substance P (S6883, MW 1347.63) and LHRH (L8008, MW 1183.27), or American Peptide Co. (Sunnyvale, CA) and used without further purification. Peptides were dissolved in water and acetonitrile (50:50 v/v) with addition of 0.1% formic acid to a final concentration of ∼1 µM. Horse myoglobin was enzymatically digested with trypsin or Asp-N in separate experiments to yield a mixture of shorter peptides following standard digestion procedures.22 Briefly, protein was diluted in ammonium bicarbonate or in sodium phosphate buffer prior to addition of a (sequencing grade trypsin (Roche Catalog No. 11 521 187 001) or endoproteinase Asp-N (Roche Catalog No. 11 420 488 001). Formic acid was added to acidify the solution and stop digestion after sample incubation at ∼37 °C overnight. Fourier Transform Ion Cyclotron Resonance Mass Spectrometry. Experiments were performed with a custom-built 9.4-T FT-ICR mass spectrometer.23,24 Ions were generated in an external (16) Oh, H.; Breuker, K.; Sze, S. K.; Ge, Y.; Carpenter, B. K.; McLafferty, F. W. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 15863-15868. (17) Tsybin, Y. O.; Witt, M.; Baykut, G.; Kjeldsen, F.; Hakansson, P. Rapid Commun. Mass Spectrom. 2003, 17, 1759-1768. (18) Nair, S. S.; Nilsson, C. L.; Emmett, M. R.; Schaub, T. M.; Gowd, K. H.; Thakur, S. S.; Krishnan, K. S.; Balaram, P.; Marshall, A. G. Anal. Chem. 2006, 78, 8082-8088. (19) Leymarie, N.; Costello, C. E.; O’Connor, P. B. J. Am. Chem. Soc. 2003, 125, 8949-8958. (20) McFarland, M. A.; Chalmers, M. J.; Quinn, J. P.; Hendrickson, C. L.; Marshall, A. G. J. Am. Soc. Mass Spectrom. 2005, 16, 1060-1066. (21) Tsybin, Y. O.; Hendrickson, C. L.; Beu, S. C.; Marshall, A. G. Proc. 53rd ASMS conference on mass spectrometry and allied topics, San Antonio, TX, 5-9 June 2005. (22) Gross, M. L.; Caprioli, R. M. The encyclopedia of mass spectrometry. Biological Applications; Elsevier: New York, 2005.

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Figure 2. Overlapping isotopic clusters for radical and prime c-type ions (left, shown for c5+ ions from substance P) and z-type ions (right, shown for z5+ ions from LHRH) generated by ECD (top) and AI-ECD (bottom). Vibrational preactivation of precursor ions in AI-ECD leads to higher ion internal energy (bottom) than in ECD alone (top).

Nanomate (Advion Biosciences, Ithaca, NY) electrospray ion source at a flow rate of ∼200 nL/min, selected by a quadrupole mass filter, externally accumulated,25 and transported by radio frequency octopole ion guides to an open-ended cylindrical ICR trap, as described elsewhere.24 For ECD, a 3-mm-diameter electron beam (1-100 ms) was injected into the ICR trap followed by an electron cleanup event (100 ms).20 The cathode potential during electron injection was -(0-6) V and was kept at +10 V otherwise. Accelerating grid voltage was at +5 V during electron injection and at -200 V otherwise. For multiple-pass electron injection, the transfer octopole dc offset was -60 V. The transfer octopole dc offset was set to +60 V during electron injection only in the singlepass electron injection regime and kept at -60 V otherwise. For activated ion (AI)-ECD experiments, an IR laser beam of variable power was injected into the ICR trap for (0-100) ms followed by electron injection. To compensate for ion magnetron motion in the ICR trap, a delay was introduced prior to ECD or AI-ECD to optimize the overlap of ion and electron beams inside the ICR trap.24 Product ions were allowed to cool for 100 ms prior to frequency-sweep excitation (72-720 kHz at 150 Hz/µs) followed by broadband detection (555 ms detection period, 512 Kword data). The time-domain transient signal was baseline corrected, Hanning apodized, zero-filled, and Fourier transformed to produce a magnitude-mode frequency spectrum that was converted to a mass-to-charge ratio spectrum.26,27 External mass/charge scale (23) Hakansson, K.; Chalmers, M. J.; Quinn, J. P.; McFarland, M. A.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2003, 75, 3256-3262. (24) Tsybin, Y. O.; Hendrickson, C. L.; Beu, S. C.; Marshall, A. G. Int. J. Mass Spectrom. 2006, 255, 144-149. (25) 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. (26) Ledford, E. B., Jr.; Rempel, D. L.; Gross, M. L. Anal. Chem. 1984, 56, 2744-2748.

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calibration was employed. Data acquisition was performed by use of a Predator data station and data analysis with MIDAS 3.21 software.28 RESULTS AND DISCUSSION Hydrogen Atom Rearrangement: ECD versus Activated Ion-ECD. In ECD of peptides, N-terminal radical product ions (c-type) demonstrate a sharp decline in abundance (decreased radical/prime ion ratio) when ion internal energy is increased by IR laser activation of precursor ions (Figure 2, left). Separation of ECD products from singly charged radical species of substance P yields mainly c′- or c•-ions (e.g., c5+′ and c5+•-ions in Figure 2, left), whereas C-terminal products are neutral (except for z9+′ ions). On the other hand, ECD of the doubly charged peptide LHRH produces mainly z-type ions due to charge retention at the Arg residue near the C-terminus. Conversely, AI-ECD mass spectra of LHRH reveal an increase (relative to ECD) in z•-ions relative to z′-ions (e.g., z5+′- and z5+•-ions in Figure 2, right). For preferential z-type ion formation from doubly charged peptides, the complementary N-terminal products are neutral and not detected. We chose the c5 (substance P) and z5 (LHRH) product ions to monitor the differences between ECD and AI-ECD, because both radical and prime components could be observed under typical ECD conditions. Other product ions from substance P and LHRH demonstrate similar behavior upon ion internal energy modulation. However, radical c-type ions or prime z-type ions are not observed by ECD or AI-ECD for some cleavages. That finding supports the results of the variance analysis performed by Zubarev et al. for (27) Shi, S. D. H.; Drader, J. J.; Freitas, M. A.; Hendrickson, C. L.; Marshall, A. G. Int. J. Mass Spectrom. 2000, 196, 591-598. (28) Blakney, G. T.; Robinson, D. E.; Ngan, V. L.; Kelleher, N. L.; Hendrickson, C. L.; Marshall, A. G., San Antonio, TX, 5-9 June 2005.

Figure 3. Radical/prime ion ratio for c5+ isotopic cluster of substance P and z5+ isotopic cluster of LHRH as a function of ion internal energy (controlled by vibrational activation), produced by (top) IR laser (100 ms irradiation period) and (bottom) ion-electron interactions. Solid curves represent a polynomial fit to discrete experimental data points. Note that the overlapping isotopic distributions of radical and prime ions were not deconvolved for the relative abundance calculations.

radical/prime product ion ratio dependence on the nature of amino acids near the cleavage site.9 The variance analysis demonstrates that the identity of the amino acid on the C-terminal side of the cleavage site strongly influences the abundance ratio of radical/ prime z-type ions. Ion Activation: Infrared Irradiation versus Energetic Electrons. Increase in ion internal energy due to increase in IR laser power in AI-ECD demonstrates monotonic and sharp decline in radical/prime c-type product ion abundance ratio for substance P and, as expected from the results shown in Figure 2, an increase of radical/prime z-type product ion ratio for LHRH (Figure 3, top). These results resemble our previously reported decrease in radical/prime c-type product ion abundance ratio for substance P for single-pass electron injection relative to the commonly applied multiple-pass regime.20,21 As previously suggested, electrons have higher energy in the single-pass regime relative to multiple pass. Therefore, vibrational activation of precursor ions may also be produced by energetic electrons. Electron kinetic energy transfer into ion internal energy may occur as a result of Coulombic interaction between an electron and an ion prior to electron capture into a high-lying Rydberg state.12 Figure 3, bottom demonstrates the systematic dependence of radical/prime ratio for c-type ions (substance P) and z-type ions (LHRH) as a function of electron energy (estimated from the cathode potential). The main point is the general trend of radical/prime ratio decline for

N-terminal product ions and increase for the C-terminal product ions. The shapes of the profile for ion internal energy variation by vibrational activation by IR photons and 0-6 eV electrons differ, especially for the N-terminal product ions. Activation with photons results in an almost exponential decay, whereas energetic-electron activation leads to a slower rate decrease of radical/prime ratio for N-terminal product ions. Presumably, increasing electron energy leads to not only higher collision energy between electrons and ions but also decreased cross section for electron capture, as reported earlier,29 whereas ion excitation by photons in the IR range derives from direct vibrational activation of precursor ions. Furthermore, wide electron energy distribution (∼1 eV at low cathode potentials, -1-2 V, and more at higher negative cathode potentials, -5-10 V)30 increases the dispersion of ion internal energy in the radical intermediate ion cloud. Note that the electron energy distribution likely extends to very low-energy values, close to 0.2 eV, even for high negative cathode potential, due to the multiple-pass method of electron injection.20 The shapes of the profiles compared between N-terminal (c-type product ions) and C-terminal (z-type product ions) for the same ion activation method should be considered by taking into account the inability to resolve the 13C12Cn-1 form of a radical ion from the monoisotopic (12Cn) peak of a prime ion in the targeted analysis performed in the broadband detection mode at 9.4 T. The isotopic correction (deconvolution of the overlapping isotopic distributions of radical and prime ions) is not included in the data representation in Figure 3, and its consideration could further improve the method reliability. Effect of Lifetime of the [c′ + z•] Complex on Final Product Ion Abundances. Following the current ECD model for product ion formation (Figure 1), the above results may be rationalized by linking ion internal energy, [c′ + z•] complex lifetime, and rate of hydrogen atom transfer between ECD products. Increased ion internal energy shortens the complex lifetime and reduces the probability of hydrogen atom transfer.9 Decreasing ion internal energy below the typical experimental temperature (cooling the ions) prevents tightly bound products from [c′ + z•]complex separation, as shown by Heeren and co-workers.31 As a result, those product ions are not detected by mass spectrometry.31 A similar effect should apply in double-resonance ECD experiments11 and in the ion trap mass spectrometers under typical ETD conditions.32,33 Indeed, a qualitative correlation can be seen for the longlived radical intermediates of substance P investigated by both double-resonance ECD34 and ETD with supplemental activation.33 Both methods support cold ECD measurements and demonstrate that the lifetimes of the radical intermediates from the N-terminus of substance P (primarily hydrophilic amino acids) are longer than (29) 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. (30) Tsybin, Y. O.; Witt, M.; Baykut, G.; Hakansson, P. Rapid Commun. Mass Spectrom. 2004, 18, 1607-1613. (31) Mihalca, R.; Kleinnijenhuis, A. J.; McDonnell, L. A.; Heck, A. J. R.; Heeren, R. M. A. J. Am. Soc. Mass Spectrom. 2004, 15, 1869-1873. (32) Swaney, D. L.; McAlister, G. C.; Wirtala, M.; Schwartz, J. C.; Syka, J. E. P.; Coon, J. J. Anal. Chem. 2007, 79, 477-485. (33) Tsybin, Y. O.; He, H.; Ben Hamidane, H.; Emmett, M. R.; Hendrickson, C. L.; Tsybin, O. Y.; Marshall, A. G., Proc. 55th ASMS conference on mass spectrometry and allied topics, Indianapolis, IN, 3-7 June 2007. (34) Cournoyer, J. J.; Lin, C.; O’Connor, P. B., Proc. 55th ASMS conference on mass spectrometry and allied topics, Indianapolis, IN, 3-7 June 2007.

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Figure 4. Application of the present product ion type determination method to myoglobin tryptic fragment [1-16]. Identification of product ion type (z- or c-type) allows for distinction between C-terminal ions (z+/z+′ increases on activation) and N-terminal ions (c+/c+′ decreases on activation) for simplified sequencing of peptides.

the lifetimes of the radical intermediates from C-terminus of substance P (primarily hydrophobic amino acids). Contrary to typical ECD results, high radical/prime ratio of c-type ions originated from short lifetime radical intermediates is observed in ETD of substance P, presumably due to lower ion internal energy in ion trap MS versus FT-ICR MS.33 Subsequent isolation and mild vibrational activation of singly charged substance P radical intermediate further increases the radical/prime ratio for these product ions. Therefore, we suggest that the final isotopic clusters formed at very low ion internal energy in ECD should demonstrate, if detected, an increased number of c•- and z′-type ions. Distinguishing N-Terminal from C-Terminal Ions: Implications for Peptide Sequencing. The opposite effect of ion activation on radical/prime ratio for N-terminal and C-terminal ECD product ions (Figures 2 and 3) suggests that ECD and AIECD could potentially distinguish between N-terminal (c-type) and C-terminal (z-type) product ions. Validation of that hypothesis was tested for a set of 10 peptides, including myoglobin enzymatic fragments and standard peptides (treated as unknowns), and demonstrated a strong potential for practical applicability. Figure 4 shows ECD and AI-ECD isotopic clusters for horse myoglobin tryptic fragment [1-16]. All displayed product ions were correctly identified as either c-type or z-type, thereby facilitating further peptide sequence tag identification. Knowing the direction sense (N-terminal or C-terminal product ions) of the sequence tag will aid in correct peptide sequence assignment, reducing the rate of false positives in protein identification. 7600

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In the present approach, bottom-up protein identification in proteomics is based on obtaining two consecutive tandem mass spectra from a target enzymatic fragment: a low-energy ECD mass spectrum followed by an activated ion-ECD mass spectrum with low-power ion excitation by IR laser irradiation (Figure 5). The radical/prime ion abundance ratio for each isotopic cluster in two tandem mass spectra enables distinction between N-terminal and C-terminal product ions, thereby facilitating subsequent peptide sequencing and database searching. Therefore, we expect this method to improve protein identification scores obtained with complementary ECD and CID techniques by taking into account ECD cleavages that are not present in CID data and thus cannot be treated with the golden pair rules and complementing the ECD/CID approach in dealing with radical c-ions. Meng et al.35 have introduced a quantitative measure for probability-based retrieval of a database protein. In the absence of knowledge of whether a fragment ion is N-terminal or Cterminal, their probability, P, of a random protein match is given by

Pf,n ) (mf )n × e-mf/n!

(1a)

m ) 4Ma/111.1

(1b)

in which 111.1 is the mass of an average amino acid, f is the (35) Meng, F. Y.; Cargile, B. J.; Miller, L. M.; Forbes, A. J.; Johnson, J. R.; Kelleher, N. L. Nat. Biotechnol. 2001, 19, 952-957.

Figure 5. Bottom-up peptide sequencing and protein identification strategy: product ion type assignment based on combined ECD and AIECD MS/MS data, to facilitate automated interpretation of ECD mass spectra in peptide analysis.

number of input fragment ions, n is the number of (spurious) matches, Ma is the mass accuracy, and posttranslational modifications are not included. If N- and C-terminal ions can be distinguished, eq 1b improves by a factor of 2:

m ) 2Ma/111.1

(2)

Thus, the confidence in identification of a protein can improve by up to several orders of magnitude, depending on the other probability parameters. We shall consider such comparisons in a future article. Method Validation and Limitations. Method validation based on a larger set of peptides, including its application to a-, b-, y-, x-, and w-type ions and posttranslationally modified peptides, as well as method evaluation for ETD in ion trap mass spectrometers, is currently underway. The current limitations of the method include reduced reliability for product ions with low signal/noise ratio and peptide structure dependence on the level of vibrational activation required to achieve sufficient change in product ion abundance ratio. The requirement for high-resolution separation of peaks between radical and prime isotopic clusters is under evaluation. Distinguishing the 13C112Cn-1 radical ion from the monoisotopic (12Cn) prime ion with the mass difference between H• and (13C-12C) of ∼4.45 mDa can be accomplished currently either with high magnetic field FT-ICR MS or by statistical averaging of many tandem mass spectra as demonstrated by Zubarev and co-workers.9 High resolving power (∼100 000) is also required to account for peptide deamidation that can occur during enzymatic digestion, resulting in distorted isotopic distributions (the deamidated and nondeamidated peptide distributions differ by 0.984 Da). Therefore, the deamidation effect should be taken into account in case the required resolution is not achieved.

Moreover, pronounced single and double H• loss has been reported for Thr and Ser residues, and to a lesser extent for Trp, Phe, and Tyr in the C-terminal position near the cleavage site.9 We also find possible hydrogen atom losses for Asn, Ile. and Phe, to be investigated separately. Reduced hydrogen atom transfer in activated ion-ECD can be considered as an instrumental means to account for single or double hydrogen atom loss/gain from ECD products that can negatively influence peptide sequencing and protein identification in database searches. CONCLUSIONS In radical peptide chemistry during ECD, c- and z-type radical and prime product ion formation is shown to depend on ion internal energy, modulated by vibrational activation of precursor ions. On average, the ratio of radical/prime c-type product ions in ECD decreases with increased ion internal energy (shorter lifetime of a radical intermediate); conversely, the ratio increases with increased ion internal energy for z-type ions. Ion interaction with energetic electrons (0-6 eV) indicates that the hydrogen atom transfer process (radical/prime ratio) depends on both average electron energy and electron energy distribution. The understanding of radical/prime ECD product ion ratio dependence on amino acid identity is not currently sufficient to provide either a qualitative or quantitative description. The hypothesis of amino acid hydrophobicity correlation with [c′ + z•]complex lifetime and, subsequently, radical/prime ratio33,36 is currently under investigation. Dependence of ECD product ion abundance (radical and prime) on ion internal energy allows for product ion type (36) Tsybin, Y. O.; He, H.; Emmett, M. R.; Hendrickson, C. L.; Marshall, A. G. Mol. Cell. Proteomics 2006, 5, S269-S269.

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(N-terminal or C-terminal) determination in many cases, based on comparison of ECD and AI-ECD mass spectra. Information on sequence tag direction from this approach should complement the alternative combination of ECD and CID to aid in peptide sequencing for protein identification and de novo peptide sequencing in tandem mass spectrometry-based proteomics. Detection of abundant c•-ions in ECD of peptides (bottom-up approach) and not in ECD of proteins and large peptides (topdown approach) supports the application of the present AI-ECD methodology in bottom-up mass spectrometry. Finally, AI-ECD can also serve as the instrumental ability to control the ratio of radical/prime ions to simplify tandem mass spectral data and increase the signal/noise ratio of a specific product ion type to facilitate automated interpretation of ECD mass spectra in peptide analysis.

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ACKNOWLEDGMENT The authors thank Sasa Kazazic for providing the enzymatic digest of myoglobin. Oleg Tsybin, Peter O’Connor, and Roman Zubarev are acknowledged for motivating discussions. We also thank Neil Kelleher and Leonid Zamdborg for identifying the improvement factor for protein search probability (eqs 1 and 2). This work was supported by the EPFL startup funds to YOT, the NSF National High-Field FT-ICR Mass Spectrometry Facility (DMR-00-84173), Florida State University, and the National High Magnetic Field Laboratory at Tallahassee, FL.

Received for review June 1, 2007. Accepted August 2, 2007. AC071165U