Activated-Electron Photodetachment Dissociation for the Structural

Sep 23, 2009 - IR and UV Photodissociation as Analytical Tools for Characterizing Lipid A Structures. James A. Madsen , Thomas W. Cullen , M. Stephen ...
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Anal. Chem. 2009, 81, 8410–8416

Activated-Electron Photodetachment Dissociation for the Structural Characterization of Protein Polyanions Vincent Larraillet,† Rodolphe Antoine,† Philippe Dugourd,† and Je´roˆme Lemoine*,‡ Universite´ de Lyon, F-69622, Lyon, France, Universite´ Lyon 1, Villeurbanne; CNRS, UMR 5579, LASIM, and Universite´ de Lyon, F-69622, Lyon, France, Universite´ Lyon 1, Villeurbanne; CNRS, UMR 5180, Sciences Analytiques Multiply deprotonated anions [M - nH]n-of large peptide mellitin, ubiquitin, and β-casein proteins were subjected to laser irradiation at 260 nm in a quadrupole ion trap. For all compounds, the predominant event consecutive to laser irradiation was the detachment of an electron. The subsequent isolation and collisional activation of the oxidized [M - nH](n-1)-• resulted in extensive fragmentation of the peptide backbone. For mellitin peptide, nearly a complete series of c•, z, and a•, x product ions were observed. Applied to proteins, this technique, coined as activated-electron photodetachment dissociation (activated-EPD), achieved much more extensive sequence coverage than regular collision activated dissociation (CAD) on the even-electron components. Furthermore, the activated-EPD spectrum of β-casein displayed phosphorylated fragment ions which suggest that the method is able to preserve part of the labile bonds of post-translational modifications. Activated-EPD is, therefore, a promising complementary technique to other dissociation techniques governed by radicals, i.e., electron capture dissociation (ECD), electron transfer dissociation (ETD), and electron detachment dissociation (EDD), for the structural characterization of large peptides and small proteins. Over the last two decades mass spectrometry has revolutionized the structural analysis of biomolecules. It has even become the cornerstone of proteomics and systems biology since it not only allows the identification and quantitation of proteins in complex mixtures but also may detect and delineate posttranslational modifications or genetic variations. Currently, two complementary strategies are applied, namely the bottom-up and top-down approaches. In the bottom-up strategy,1 proteins are subjected to enzymatic hydrolysis and the resulting peptides are ionized by matrix-assisted laser desorption ionization (MALDI) or electrospray ionization (ESI). Peptide ions are mass analyzed or may be subjected to low energy collision activated dissociation tandem mass spectrometry analysis (CAD-MS/MS), which induces predominant backbone fragmentations throughout the amide bonds. The pattern of fragment ions provides information * Corresponding author. E-mail: [email protected]. † CNRS, UMR5579, LASIM. ‡ CNRS, UMR 5180, Sciences Analytiques. (1) Chait, B. T. Science 2006, 314, 65.

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on the peptide sequence and, in the favorable cases where they are not preferentially cleaved, on the location of post-translational modifications. Although it is by far the most common technology used for routine and large scale identification of proteins in complex mixtures, the bottom-up approach often fails to detect or distinguish between the genetic or post-translationally modified isoforms of a protein, as typically no more than 50% of the protein sequence is covered. Conversely, the top-down approach2 is based on the gas phase fragmentation of protonated molecular ions of intact proteins. Though preliminary experiments have shown that peptide sequence tags from whole proteins are accessible by CAD,3 IRMPD (infrared multiphoton dissociation),4 or BIRD (blackbody infrared radiative dissociation)5 activation modes, the concept of top-down protein identification really emerged, thanks to the decisive development of electron capture dissociation (ECD).6,7 ECD may take place in the Penning trap of a Fourier transform ion cyclotron resonance mass spectrometer (FTICR) where confined multiply protonated proteins react with thermal electrons to create a hypervalent species (R-NH3•). The high recombination energy (∼5 eV) of an electron with the protonated species diminishes the influence of peptide bond dissociation energies and preferential cleavages. Therefore, ECD results in a marked improvement in the extent of inter-residue backbone cleavages. In order to extend top down identification of proteins up to 50 kDa, ECD may be combined with earlier or concurrent CADor IRMPD-catalyzed disruption of the stabilizing weak bonds of the tertiary structure.8 Hunt and co-workers introduced the electron transfer dissociation (ETD) technique9 in an effort to (2) (a) Chen, C.-H. W. Anal. Chim. Acta 2008, 624, 16–36. (b) Kelleher, N. L. Anal. Chem. 2004, 76, 197A. (c) Bogdanov, B.; Smith, R. D. Mass Spectrom. Rev. 2005, 24, 168–200. (3) Nemeth-Cawley, J. F.; Tangarone, B. S.; Rouse, J. C. J. Proteome Res. 2003, 2, 495–505. (4) Raspopov, S. A.; El-Faramawy, A.; Thomson, B. A.; Siu, K. W. M. Anal. Chem. 2006, 78, 4572–4577. (5) Ge, Y.; Horn, D. M.; McLafferty, F. W. Int. J. Mass Spectrom. 2001, 210, 203–214. (6) (a) Zubarev, R. A. Curr. Opin. Biotech. 2004, 15, 12–16. (b) McLafferty, F. W.; Horn, D. M.; Breuker, K.; Ge, Y.; Lewis, M. A.; Cerda, B.; Zubarev, R. A.; Carpenter, B. K. J. Am. Soc. Mass Spectrom. 2001, 12, 245–249. (c) Cooper, H. J.; Hakansson, K.; Marshall, A. G. Mass Spectrom. Rev. 2005, 24, 201–222. (7) Shi, S. D. H.; Hemling, M. E.; Carr, S. A.; Horn, D. M.; Lindh, I.; McLafferty, F. W. Anal. Chem. 2001, 73, 19–22. (8) Horn, D. M.; Ge, Y.; McLafferty, F. W. Anal. Chem. 2000, 72, 4778–4784. (9) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. 2004, 101, 9528–9533. 10.1021/ac901304d CCC: $40.75  2009 American Chemical Society Published on Web 09/23/2009

Figure 1. Schematic diagram of the experimental setup.

implement an analogous radical-induced fragmentation pathway in the widely available quadrupole ion trap instrument (QIT). Employing an ion/ion reaction, ETD initiates protein and large peptide dissociation by transferring an electron from a radical anion to a multiply protonated molecular ion. Fragmentations of the N-CR bond of the peptide backbone create complementary c and z ion type series of fragments, as does ECD. Though current QIT have limited m/z ranges and resolutions, ETD spectra are usually informative enough for simple protein identification. Optical excitation with high energy photons in the UV range is another way to generate reactive radicals and bypass the preferential cleavages observed at low collision energy in CAD mode. In the case of peptides, nearly complete peptide sequence coverage has proved to be accessible by laser photodissociation at 193 nm10 and 157 nm.11 These two wavelengths are close to amide absorption bands and consequently result in excitation throughout the molecule. Alternatively, more specific excitation of peptide side chain chromophores occurs above 220 nm, which opened unprecedented fragmentation pathways of protonated molecular ions.12 Similarly, radical peptides can be produced by cleaving molecular metal complexes.13 Julian and co-workers highlighted backbone specific cleavages of protonated whole proteins after laser irradiation at 266 nm of iodinated tyrosyl and histidyl residues and subsequent collisional activation of the isolated radical species.14 Furthermore, recent works have revealed that irradiation around 260 nm of peptides15 or DNA polyanions (10) Williams, E. R.; Furlong, J. P. P.; McLafferty, F. W. J. Am. Soc. Mass Spectrom. 1990, 1, 288. (11) Thompson, M. S.; Cui, W. D.; Reilly, J. P. Angew. Chem., Int. Ed. 2004, 43, 4791–4794. (12) (a) Tabarin, T.; Antoine, R.; Broyer, M.; Dugourd, P. Rapid Commun. Mass Spectrom. 2005, 19, 2883–2892. (b) Lemoine, J.; Tabarin, T.; Antoine, R.; Broyer, M.; Dugourd, P. Rapid Commun. Mass Spectrom. 2006, 20, 507– 511. (c) Wilson, J. J.; Brodbelt, J. S. Anal. Chem. 2008, 80, 5186-5196; Wilson, J. J.; Brodbelt, J. S. Anal. Chem. 2007, 79, 7883–7892. (13) (a) Laskin, J.; Yang, Z. B.; Lam, C.; Chu, I. K. Anal. Chem. 2007, 79, 6607– 6614. (b) Chu, I. K.; Rodriguez, C. F.; Rodriguez, F.; Hopkinson, A. C.; Siu, K. W. M. J. Am. Soc. Mass Spectrom. 2001, 12, 1114–1119. (c) Barlow, C. K.; McFadyen, W. D.; O’Hair, R. A. J. J. Am. Chem. Soc. 2005, 127, 6109–6115. (14) Ly, T.; Julian, R. R. J. Am. Chem. Soc. 2008, 130, 351–358. (15) (a) Joly, L.; Antoine, R.; Allouche, A.-R.; Broyer, M.; Lemoine, J.; Dugourd, P. J. Am. Chem. Soc. 2007, 129, 8428. (b) Antoine, R.; Broyer, M.; ChamotRooke, J.; Dedonder, C.; Desfranc¸ois, C.; Dugourd, P.; Gre´goire, G.; Jouvet, C.; Onidas, D.; Poulain, P.; Tabarin, T.; van der Rest, G. Rapid Commun. Mass Spectrom. 2006, 20, 1648–1652.

causes electron photodetachment.16 The subsequent collisional activation of the isolated radical anions of small peptides or phosphorylated peptides,17 a technique we coined activated electron photodetachment dissociation (activated-EPD), gave rise to intense series of a and x fragment ions similar to those produced by electron detachment dissociation (EDD) and reverse-electron transfer dissociation (ETD) on polyanions of peptides.18-20 EDD and reverse-ETD techniques involve the bombardment of polyanions with electrons or charge transfer with radical cations. Herein, we investigate for the first time the merits of activatedEPD for the dissociation of polyanions of large peptides or intact proteins. Compared to direct CAD of even-electron species, activated-EPD leads to much more intense and extensive fragmentations of the peptide backbone and opens a new perspective in the field of structural characterization and top-down sequencing of large peptides and proteins. EXPERIMENTAL SECTION Chemicals. Peptides and proteins were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Mellitin, ubiquitin, and β-casein were dissolved in 50/50 water/acetonitrile (v/ v) at a concentration of 100 µM and directly electrosprayed. Sources conditions were optimized to produce high-charge states of protein polyanions. Mass Spectrometry. Activated-EPD experiments were performed using a modified linear quadrupole ion trap mass spectrometer (LTQ, Thermo Fisher Scientific, San Jose, CA) with enlargement for the high 2000-4000 Th range. A quartz window was fitted on the rear of the LTQ chamber to allow the introduction of a UV laser beam (see Figure 1). The laser is a nanosecond frequency-doubled tunable Panther EX OPO laser pumped by a Surelite II Nd:YAG laser (both from Continuum, Santa Clara, (16) (a) Gabelica, V.; Rosu, F.; Tabarin, T.; Kinet, C.; Antoine, R.; Broyer, M.; De Pauw, E.; Dugourd, P. J. Am. Chem. Soc. 2007, 129, 4706. (b) Gabelica, V.; Tabarin, T.; Antoine, R.; Rosu, F.; Compagnon, I.; Broyer, M.; De Pauw, E.; Dugourd, P. Anal. Chem. 2006, 78, 6564. (17) Antoine, R.; Joly, L.; Tabarin, T.; Broyer, M.; Dugourd, P.; Lemoine, J. Rapid Commun. Mass Spectrom. 2007, 21, 265. (18) Coon, J. J.; Shabanowitz, J.; Hunt, D. F.; Syka, J. E. P. J. Am. Soc. Mass Spectrom. 2005, 16, 880–882. (19) Kjeldsen, F.; Silivra, O. A.; Ivonin, I. A.; Haselmann, K. F.; Gorshkov, M.; Zubarev, R. A. Chem.sEur. J. 2005, 11, 1803–1812. (20) Budnik, B.; Haselmann, K.; Zubarev, R. Chem. Phys. Lett. 2001, 342, 299– 302.

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CA). All the activated-EPD experiments were performed at λ ) 260 nm. The output power for this wavelength was ∼10 mW at a repetition rate of 10 Hz. The laser beam passes through two diaphragms (2 mm diameter), lenses, and a mechanical shutter electronically synchronized with the mass spectrometer, after which it is injected on the axis of the linear trap. The mechanical shutter is used to synchronize the laser irradiation with the trapping of the ions. To perform laser irradiation for a given number of laser pulses, we add in the ion trap RF sequence an MSn step with an activation amplitude of 0%, during which the shutter located on the laser beam is opened. A half wave plate and a polarizer are included in the laser path to control the laser power. The laser power is monitored in front of the mass spectrometer by a power meter (OphirSpiricon GmbH, Ahrensburg, Germany). The maximum laser energy that enters the trap is ∼600 µJ/pulse. Collision induced dissociation (CAD) experiments were performed with the same apparatus. This was accomplished using helium gas at a normalized collision energy of 15% for 30 ms. The activation q value was set to 0.25 for CAD and for activated-EPD (0.21 for mellitin). An m/z window of 3 Th was applied for precursor ion isolation for both methods. The proteins were electrosprayed at a flow rate of 5 µL min-1 in negative-ion mode. For the activated-EPD experiments, mellitin and ubiquitin were irradiated with five laser shots while β-casein was subjected to a single shot. The activated-EPD and direct CAD fragment ions were assigned according to predicted fragmentation peak lists provided by ProteinProspector (v. 5.2.2). Experimental peaks were assigned using zoom and ultra zoom scans (mass resolution 30 000, mass accuracy 0.2 Th) and simulated isotopic distributions. RESULTS AND DISCUSSION As illustrated by eq 1 below, the activated-EPD method consists of an initial step of UV irradiation of a multiple deprotonated anion.17,21 The main consequence of UV irradiation of such species is the loss of one electron resulting in an oxidized form of the precursor ion. In the case of peptides, the subsequent collisional activation of the isolated radical anion results in intense cleavages of the backbone, whereas only small neutral losses were observed when CAD was applied to even-electron precursor ions. hν

(n-1)-•

[M - nH]n- 98 [M - nH]

CAD

98 fragments

(1)

Activated-EPD of Peptide Mellitin. First, the doubly deprotonated [M - 2H]2- molecular ion of mellitin, a 26 mer peptide of molecular weight 2846, was isolated and irradiated at 260 nm by UV laser with five laser shots to produce the radical [M - 2H]-• ion (see Figure 2A). The inset shows the plot of the ratio between the summed intensities of the fragment species (oxidized anion and minor fragment peaks) and the intensity of the [M - 2H]2- molecular ion relative to the laser power. The linear relation of the electron detachment yield toward the laser power is in agreement with a one-photon detachment process and confirms previous reports.17 At the highest power used in (21) Gabelica, V.; Tabarin, T.; Antoine, R.; Rosu, F.; Compagnon, I.; Broyer, M.; De Pauw, E.; Dugourd, P. Anal. Chem. 2006, 78, 6564–6572.

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this experiment, the yield of electron detachment is higher than 90%. As mentioned above, fragment ions are noticeable on the electron photodetachment spectrum but their relative intensities are too weak to cover the peptide sequence extensively. In contrast, the activated-EPD mass spectrum obtained after isolation and collisional heating of the oxidized species [M 2H]- · of mellitin displays intense fragment ions (Figure 2B). Neutral losses of 18 and 44 Da are observed and are, respectively, attributed to H2O and CO2 losses. The intense loss of CO2, while not observed on the CAD spectrum of the even-electron [M 2H]-, is probably due to direct homolytic recombination of a carboxylate radical after electron detachment at the C terminus carboxyl group.22 More information is obtained from the nearly complete series of product ions attributed to peptide backbone cleavages. Taking into account the experimental m/z values of the most intense fragment ions (Figure 2B) indicates that activated-EPD occurs through two prominent pathways leading to complementary c and z type ions and complementary a and x product ions series following N-CR and CR-C bond cleavages, respectively. To assess the electron parity across the pattern of activated-EPD product ions, the experimental isotopic distribution of each fragment ion recorded with the high resolution mode of the quadrupole ion trap (ultra zoom scan) was matched to its simulated isotopic distribution. This comparison evidence is a radical state for most c and a type fragment ions while their complementary z and x species are even-electron components. Note that a definitive proof could be brought by the implementation of the activated-EPD experiment on an FTICR instrument in order to achieve exact mass measurements. The observed oddelectron a and even-electron x ion fragments are consistent with the fragmentation feature of EDD pattern of peptides occurring through proximal recombination of the radical site (Figure 3B).19,23 While regular ECD fragmentation mechanism favors even-electron c fragment ions with highly stabilizing amide structure,24 the activated-EPD spectrum displays an intense oddelectron c18 product ion. This dominating fragment corresponds to cleavage on the N-terminal side of the tryptophan residue where the electronic excitation is expected to be initially localized after a resonant laser excitation at 260 nm. From a mechanistic point of view, the intense c18-• fragment ion could originate from the initial formation of a cationic radical on the indolic side group of tryptophan residue. The subsequent recombination across the adjacent N-CR bond releases predominant c18-• and complementary z8 product ions (see Figure 3A). At this stage, however, the activated-EPD fragmentation mechanisms remain speculative. For instance, the influence of collisional heating prior to or after the laser irradiation need to be further investigated toward the relative abundance and electron parity of activated-EPD product ions. Indeed, previous studies have illustrated how increasing the initial ion energy prior to ECD or subsequently to ETD markedly modified the relative intensities (22) Antoine, R.; Joly, L.; Allouche, A.-R.; Broyer, M.; Lemoine, J.; Dugourd, P. Eur. Phys. J. D 2009, 51, 117. (23) Anusiewicz, I.; Jasionowski, M.; Skurski, P.; Simons, J. J. Phys. Chem. A 2006, 109, 11332–11337. (24) Frison, G.; van der Rest, G.; Turecek, F.; Besson, T.; Lemaire, J.; Maitre, P.; Chamot-Rooke, J. J. Am. Chem. Soc. 2008, 130, 14916.

Figure 2. (A) Photodissociation spectrum of isolated [M - 2H]2- ion of mellitin under five laser shots at 260 nm. The inset plots the measured efficiency of electron photodetachment as a function of the laser power. (B, C) Activated-electron photodetachment dissociation spectra (300 scans) of oxidized and isolated [M - 2H]2-•. ion of mellitin under a normalized CAD collision energy of 15% for 30 ms.

between odd c•, z• and even c, z product ions25 compared to regular ECD and ETD. Overall, 24 of 25 possible inter-residue bonds were seen to be cleaved, enabling the full sequence coverage of mellitin peptide (96%). Whereas the CAD is carried out on the even-electron species [M - H]-, only 54% of the peptide sequence is covered by the pattern of b, y, and a type fragments ions. This striking (25) (a) Tsybin, Y. O.; He, H.; Emmett, M. R.; Hendrickson, C. L.; Marshall, A. G. Anal. Chem. 2007, 79, 7596. (b) Ben Hichame, H.; Chiappe, D.; Hartmer, R.; Vorobyev, A.; Moniatte, M.; Tsybin, Y. O. J. Am. Soc. Mass Spectrom. 2009, 20, 567.

contrast between activated-EPD and CAD further illustrates the general trend of increased efficiency of dissociation of radical ions as observed for methods involving electron excitations and charge transfer.18,19,26 Activated-EPD on Proteins. In order to evaluate the ability of activated-EPD in providing sequence information from intact proteins, we first studied the ubiquitin protein model of 8.6 kDa. The negative electrospray spectrum of ubiquitin displays a charge (26) 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.

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Figure 3. Proposed fragmentation mechanisms supporting the a•, c•, x, and z product ions observed on an activated-electron photodetachment dissociation spectrum of mellitin.

distribution ranging from 6 to 12 centered (data not shown) on the most intense [M - 8H]8- anion species. Five shots of UV irradiation of the isolated [M - 8H]8- precursor ions of ubiquitin give rise to the oxidized radical [M - 8H]7-• species with a yield of 26%. This radical anion was then isolated and subjected to collisional excitation. The activated-EPD spectrum is shown in Figure 4 and displays a much more complex fragmentation pattern than does the CAD one (data not shown). Numerous product ions have an m/z value above the precursor ion m/z value at 1222, consistent with a lower charge state comprising a dissociation process accompanied by charge loss. Indeed, zooming on restricted areas of the activated-EPD spectrum reveals that most of the isotopic clusters are characteristic of charge states ranging from 1- to 6-. The fragmentation pattern identified from the peak list predicted by Protein Prospector is given for the ubiquitin sequence (Figure 4B). It reveals a marked improvement of peptide sequence coverage (58%) over that obtained by CAD of the even-electron [M - 7H]7- precursor ion (21%) (Figure 4C). Activated-EPD was finally applied to β-casein, a protein of 24.05 kDa phosphorylated on five serine residues. Figure 5A displays the negative electrospray spectrum and a wide distribution of multiply deprotonated anions with charge states ranging from 23to 9-. Activated-EPD was carried out on the most intense charge state 16-. The isolated [M - 16H]16- ion was isolated and irradiated with one laser shot leading to the single [M 16H]15-•. Figure 5B compares CAD and activated-EPD spectra recorded respectively on [M - 15H]15- and [M - 16H]15-• with the same activation time and energy (30 ms and 13%). As observed in the case of ubiquitin, the activated-EPD spectrum depicts an increased dissociation yield and more complex fragmentation pattern with regard to the CAD spectrum. However, an overall decrease in the yield of fragmentation is observed in comparison to the smaller ubiquitin protein, presumably due to its higher protein size. This feature, dependent on protein size, has also been noticed during ECD and was attributed to strengthening of intramolecular interactions after water removal in the electrospray interface.8 This 8414

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conformational stabilization may be drastically reduced by “heating” the folded ions with CAD or IRMPD prior to ECD. Among the few peaks attributed in the low m/z region, owing to their reduced charge state, four a type product ions were assigned as phosphorylated fragments bearing, respectively, one phosphate group for a29, a30, and a44 product ions and four phosphate groups for a27 components. This feature indicates that activatedEPD of radical polyanions is mild enough to keep intact the labile bonds between the serine or threonine side chains and the phosphate groups. Phosphorylation of serine and threonine is one of the most common post-translational modifications (PTM) of proteins and positioning of this modification along the peptide chain is critical owing to its critical role in the control of essential biological events. Numerous studies have already illustrated the great interest of ECD and ETD and, more recently, activated-EPD methods to preserve from cleavage the weak bond between numerous post-translational modifications such as phosphorylation, glycosylation, sulfatation, nitrosylation, and the peptide chain.7,27 However, in the case of large proteins, the higher-order structure of gaseous ions directs the fragmentation at the peptide bonds preferentially over PTM removal.28 Hence, our above observation of phosphorylated fragment ions prompts us to perform a more systematic study on whether activated-EPD could share with ECD or ETD similar potential for positioning different types of PTM on small proteins or peptides. Concerning the highly charged product ions, the limited resolution and mass accuracy of the linear quadrupole ion trap prevented the unambiguous assignment of product ions with high charge states for which the isotopic distributions are not resolved or correspond to overlapping fragments. Hence, we were not able to conclude on the number of inter-residue bond cleavages. This limitation could be overcome by ion/ion reaction-driven charge reduction leading to either singly charged ions29 or partially reduced species30 that may then be converted to a zero charge spectrum after deconvolution.31 In the case of activated-EPD carried out on polyanions, charge reduction should occur after proton transfer from a protonated donor with sufficient low proton affinity. Another obvious alternative would be the implementation of activated-EPD within mass spectrometers of high resolution capacity such as hybrid quadrupole-FTICR or orbitrap instruments. A more general question concerns how the implementation of activated-EPD could be profitable in proteomic laboratories. Though it is obviously too early to claim that activated-EPD in a QIT is competitive with ECD for true top down purposes of very large proteins, we argue that it is already sufficiently efficient for the structural characterization of large and potentially modified large peptides or proteins below 10 kDa. Activated-EPD is also an interesting alternative when the ETD option is not available on an existing quadrupole ion trap instrument, since its imple(27) (a) Mirgorodskaya, E.; Roepstorff, P.; Zubarev, R. A. Anal. Chem. 1999, 71, 4431–4436. (b) 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. (c) Chi, A.; Huttenhower, C.; Geer, L. Y.; Coon, J. J.; Syka, J. E. P.; Bai, D. L.; Shabanowitz, J.; Burke, D. J.; Troyanskaya, O. G.; Hunt, D. F Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 2193–2198. (28) Siuti, N.; Kelleher, N. L. Nat. Methods 2007, 4, 817. (29) McLuckey, S. A.; Stephenson, J. L.; Asano, K. G. Anal. Chem. 1998, 70, 1198–1202. (30) Scalf, M.; Westphall, M. S.; Smith, L. M. Anal. Chem. 2000, 72, 52–60. (31) Stephenson, J. L.; McLuckey, S. A. J. Mass Spectrom. 1998, 33, 664–672.

Figure 4. (A) Activated-electron photodetachment dissociation spectrum of an oxidized and isolated [M - 8H]7- · ion of ubiquitin. The inset shows the photodissociation spectrum of isolated [M - 8H]8- ion of ubiquitin after five laser shots at 260 nm. (B) Peptide sequence coverage of ubiquitin deduced from the whole pattern of attributed product ions on activated-EPD spectrum. (C) Peptide sequence coverage of ubiquitin deduced from the whole pattern of attributed product ions on a regular CAD spectrum performed on [M - 7H]7- molecular ion. (D) Partial views of activated-EPD spectrum of oxidized and isolated [M - 8H]7- · ion of ubiquitin.

mentation does not require major hardware modification or upgrading in the case of linear ion trap hardware.32 The sample concentrations and time of acquisition required for the presented data obviously does not compete with the current performances achieved by commercial devices operating in ECD or ETD mode; i.e., 100 µM of a protein solution was infused in a conventional flow electrospray during a 5 min acquisition time in normal mode scanning. A high repetition rate or continuous wave UV laser (32) Dugourd, P.; Antoine, R.; Broyer, M.; Talbot, F. O. Patent PCTFR2005003142, 2006, ppWO 2006/064132 A064131.

devices combined with a nanospray source will undoubtedly dramatically reduce the duty cycle and sample consumption. Note that, if needed (for precursor ions that would present low electron detachment yields), the efficiency of the electron detachment process could be increased by increasing the laser power (for example, by the use of the fourth harmonics of the Nd:YAG laser). Since activated-EPD is really efficient for peptide sequence coverage in the 5 to 10 kDa mass range, it is also possible to envisage of developing protein mixture profiling after limited proteolysis degradation and off-line coupling with liquid chromaAnalytical Chemistry, Vol. 81, No. 20, October 15, 2009

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Figure 5. (A) Full electrospray mass spectrum of β-casein in negative mode. The inset shows the photodissociation spectrum of isolated [M - 16H]16- ion of β-casein after one laser shot at 260 nm. (B) Comparison of activated electron photodetachment dissociation spectrum of an oxidized and isolated [M - 16H]15-• ion (positive values) and regular CAD of [M - 15H]15- ion (negative values) of β-casein at a normalized CAD collision energy of 13% for 30 ms. (C) Partial view over 900-1400 Th. * indicates the number of phosphate groups retained by the fragment ions.

tography. Proteolytic enzymes or chemical cleavage (BrCN) giving rise to larger peptides (30 to 80 residues) may indeed replace the gold standard trypsin, which in turn could dramatically reduce the complexity of final samples. CONCLUSIONS The data presented above shows that we have described the merits of a new technology for the first time, namely activatedelectron photodetachment dissociation (activated-EPD), which has the potential to constitute a powerful alternative to existing technologies aimed at top-down sequencing of large peptides and proteins. The method is based on the generation of radical anions by a UV laser, the isolation of the oxidized radical anions, and subsequent fragmentation initiated by collision-induced dissociation. Activated-EPD was easily implemented on a quadrupole ion trap and sequence coverage up to 96 and 58% was achieved for mellitin and ubiquitin, respectively, after assignment of the pattern of fragment ions originating from N-CR and CR-C and backbone cleavages. Such a dissociation yield is close to those observed during ECD experiments implemented in FTICR. As quadrupole 8416

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ion traps have limited mass resolution, activated-EPD carried out in such instruments is at the moment clearly more dedicated to top-down sequencing of large peptides or proteins below 15 kDa, unless the assignment ambiguities of fragment ions with high charge states are solved by a charge reduction process or implementation of activated-EPD in hybrid quadrupole-FTICR or orbitrap instrument or a simple FTICR compatible with the SORI CAD mode. ACKNOWLEDGMENT We thank Ezus Lyon I and the Institut de Chimie de Lyon (Director P. Sautet) for their financial support in acquiring the LTQ instrument and OPO pumped Nd:YAG laser. SUPPORTING INFORMATION AVAILABLE Sequence of β-casein. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review June 16, 2009. Accepted September 12, 2009. AC901304D