Mechanisms of UV Photodissociation of Small Protonated Peptides

J. Phys. Chem. A 2010, 114, 3147–3156

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Mechanisms of UV Photodissociation of Small Protonated Peptides† M. Pe´rot UniVersite´ Paris-Sud, Laboratoire des Collisions Atomiques et Molećulaires, UMR 8625, Baˆt. 351, Orsay, F-91405 France and UniVersite´. Paris-Sud, Laboratoire de Photophysique Molećulaire, UPR 3361, Baˆt. 210, Orsay, F-91405 France

B. Lucas, M. Barat, and J. A. Fayeton* UniVersite´ Paris-Sud, Laboratoire des Collisions Atomiques et Molećulaires, UMR 8625, Baˆt. 351, Orsay, F-91405 France, and CNRS, UMR 8625, Orsay, F-91405 France

C. Jouvet UniVersite´ Paris-Sud, Laboratoire de Photophysique Molećulaire, UPR 3361, Baˆt. 210, Orsay, F-91405 France, and CNRS, UPR 3361, Orsay, F-91405 France ReceiVed: September 15, 2009; ReVised Manuscript ReceiVed: NoVember 3, 2009

Photofragmentation of protonated dipeptides by 263 nm photons is investigated with an experimental technique based on the detection in coincidence of the ionic and neutral fragments. With this method, it is possible to determine whether the fragmentation takes place in one or several steps. The timing of these steps can also be evaluated. The interpretation of the various fragmentation pathways is tentatively developed along the same line as that previously proposed for tryptophan. The fragmentation can be explained by two types of mechanisms: internal conversions and direct fragmentations triggered by the migration of the photoactive electron on positive charged sites or on oxygen sites. I. Introduction 1-3

Recent studies show that photons induce specific dissociations of protonated biomolecules that are found neither in collision induced dissociation (CID) nor in electron capture dissociation (ECD). Similar findings were found in peptide fragmentation as shown by the recent work of the Lyon group.4 For example, optical excitation of the chromophore, which is in peptides one of the aromatic amino acid, such as phenylalanine, tyrosine, and tryptophan, leads to CR-Cβ fragmentation. One surprising result is that such specific fragmentation occurs in some sequences but not in some others. There is probably a link between these fragmentations and the sequence or most likely with the secondary structure of peptides. For example, fragmentation that might occur in a chain-like structure (R helix) would be inhibited in a globular structure in which the aromatic residue is kept in interaction with many other amino acids. In a previous paper5 we have investigated in detail the fragmentation of protonated tryptophan using a new experimental method in which ionic and neutral fragments produced in a same event are detected in coincidence allowing multistep fragmentation be analyzed. This technique also enables us to determine fragmentation times from nanosecond to millisecond. The conclusion of that work was that photofragmentation of protonated aromatic amino acids either results from an internal conversion (IC) to the ground state6 or is induced by direct dissociation of an excited state, the nature of which, determines the fragmentation pathway. Since the ordering of the excited states is dependent on the conformation of the amino acid, the †

Part of the “Benoît Soep Festschrift”. * Corresponding author. E-mail: [email protected].

fragmentation pathways also depend on the initial geometry as demonstrated in the tyrosine case.7 Three excited states with different electronic configurations are involved in the photodissociation of protonated tryptophan6,8 (Figure 1a). The ππ* state with the active electron localized on the indole ring bears the oscillator strength for UV excitation. Excited state optimization shows a barrier free proton transfer from the NH3+ group toward the indole ring that gives rise to the production of m/z ) 132 fragments8 after the CR-Cβ bond breaking and a subsequent hydrogen transfer from the glycine to the indole ring. In the same energy range lies a πσ*NH3 state resulting from a transfer of the active electron from the π* indole ring orbital (the indole being the symmetry plane) toward a σ*NH3 orbital located on the amino group of the protonated tryptophan. The σ*NH3 orbital has a σ symmetry with respect to the amino acid plane. This state decays via an H loss through a small barrier within 15 ps and leads to the formation of a m/z 204 radical cation that can further fragment producing a m/z 130 fragment ion at very long time.6 The third excited state lies higher in energy and can be characterized as a charge transfer state with the active electron localized on a π*CO orbital with respect to the amino-acid plane. Its decay involves a barrier free proton transfer from the NH3+ group toward the carbonyl group within 400 fs. The concerted electron-proton transfer to the carbonyl weakens the CR-Cβ bond and leads to the production of m/z 130 ionic fragments.2,8 The aim of the present paper is to study the photodissociation of four protonated dipeptides (Figure 2) using the multicoincidence technique to see if the overall scheme developed in the case of tryptophan can be extended to larger systems. The same photoactive tryptophan (W) is associated with glycine (G) the simplest amino acid, [GW]H+ and [WG]H+, and with

10.1021/jp908937s  2010 American Chemical Society Published on Web 11/20/2009

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Figure 1. (a) Protonated tryptophan. The three sites πσ*NH3, ππ*, and ππ*CO of the active electron are indicated. (b) Protonated tripeptide [GWG]H+. The nomenclatures of bond breakages (a, b, c and x, y, z17) as well as the active sites (1 to 5) involved in photofragmentation pathways (see text) are indicated.

Figure 2. Five studied molecules. The most important a1, y1, z1, a2, y2, and z2 bond breakages17 observed in this study are indicated.

leucine (L), an amino acid with a long alkyl chain [LW]H+ and [WL]H+, with the W located on the C or N terminal, respectively. This study is then applied to [GWG]H+, a tripeptide in which the tryptophan is encapsulated between two glycines. From femtosecond experiments9-12 it appears that the excited state lifetimes of all these ions are very short (about 100 fs), which implies very fast excited state dynamics as in the tryptophan case. II. Experimental Section The experimental setup described in detail in previous papers5,13 is shown in Figure 3. Protonated peptides produced by an electrospray ion source (ESI) are trapped inside an hexapole for one millisecond and then accelerated to 2.5 keV. Ion bunches of 100 ns time width produced at a 1 kHz repetition rate are mass selected by an electric chopper. The ions then enter a Zajfman ion trap,14-16 in which they can be stored for a few milliseconds. The ions are irradiated by a 263 nm light pulse of about 100 µJ and 200 ns time width, at a 1 kHz repetition rate. The laser can be fired either in the ion trap or downstream inside an interaction box that can be polarized. Before entering the polarized box, the ions are accelerated to a kinetic energy E0 ) 5000 eV. The neutral fragments are received on a first position sensitive detector (PSD) located 1 m from

the polarized region. Ionic fragments are mass selected in a 45° electrostatic analyzer and received on a second PSD. For each laser shot, arrival times and positions of neutral and ionic fragments are recorded (tn, Yn, and Zn for the neutral fragments and ti, Yi, and Zi for the ion fragment). An event is defined as a coincidence between an ion fragment and one or two neutral fragments coming from the dissociation of a given molecule and produced during one laser shot. If more than one neutral fragment is produced in a given fragmentation event, triple coincidences, one ion and two neutrals, can be recorded as long as the arrival times between the two neutrals is larger than 40 ns, a limitation due to the dead time of the PSD. The flux of incident projectiles and photons is kept low enough to record at most one event per ion pulse to avoid false coincident events. Fragmentation Times. Firing the laser inside the ion trap allows to measure long fragmentation times, typically longer than 5 µs obtained from the time spectra of neutral fragments escaping the trap.5 Fast fragmentations, t < 200 ns, can be investigated by firing the laser inside the interaction box. In such a case, the fragmentation times can be measured by establishing an axial electric field inside the box. In fact, the neutral fragment keeps the velocity it gets at the fragmentation time while the ion fragment is further decelerated. The detailed calculation of the fragmentation time is given in ref 13. The

UV Photodissociation of Small Protonated Peptides

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Figure 3. Experimental setup. Ions are irradiated with the laser fired either in the electrostatic ion beam trap (1) or in a interaction box (2).

time resolution is actually limited to 10 ns due to the size of the laser spot and the kinetic energy released. The last case corresponds to fragmentations occurring in the time window (200 ns to a few microseconds) after leaving the box. Therefore, fragmentation times can be measured in a wide scale extending over 5 decades from 10 ns to a few microseconds. “VV” Correlations. To characterize a fragmentation event, an important aspect concerns the choice of the most pertinent observables and when chosen, the most significant correlation between them. The VV correlation between the velocity of the neutral and the ion fragment is one of such very important observables since this is a way to determine the number of fragmentation steps as well as the mass of the neutral fragments. The “ZZ” contour plot has been defined in several papers (see, e.g., ref 5) as the ratio between the components along the Z axis normal to the analyzer plane (Figure 3) of Zi and Zn, the locations on the PSDs of the neutral and ion fragments. Actually in this paper this contour plot is replaced by the “VV” contour plot, the ratio between the velocity components Vi ) Zi/Ti and Vn ) Zn/Tn with Ti, Tn the times spent by the fragments from the interaction region to the detectors. We found this choice more adequate since, for a two-body dissociation, the “VV” slope directly provides the mass ratio between the neutral and the ionic fragments due to momentum conservation. Indeed, for a binary dissociation the “VV” contour plot stretched along a line for which the slope is given by the ratio of the masses of the two fragments. Some “VV” contour plots show, however, a line shape with different slope or a bulky shape. Such a pattern reflects a dissociation producing more than one neutral fragment. Information on a three-body fragmentation process can, however, still be extracted from such “VV” contour plots between the ion fragment and only one out of the two (or more) neutrals fragments. The masses of the fragments and the order of the various fragmentation steps can be deduced from the analysis of the triple coincidences. N(Yi,ti - tn) Correlations. Another correlation pattern used here (Figure 5), is the number of coincidence events N(Yi,ti tn) expressed as a function of Yi the location of the ion impact on the MCP providing the energy of the ionic fragment, and ti - tn the difference of arrival times between the ion and neutral fragments.5 This diagram allows disentangling, for a given m/z ion fragment, fast fragmentations occurring inside the interaction box polarized at a voltage V from slow fragmentations occurring downstream, between the box and the analyzer. III. Results and Discussion The relative population (table 1) of each fragmentation pathway is obtained as the ratio between the number of detected ion fragments of a given mass and the total number of neutral

fragments. Some features should be noted. The positive charge remains attached to the indole chromophore due to its low ionization potential. For example, notice the importance of a1 ion fragments in [WG]H+ and [WL]H+ while the y1 ion fragments dominate in [GW]H+ and [LW]H+ fragmentation. [GWG]H+ breaks on both sides of the chromophore, resulting in production of a2 and y2 ion fragments. The yi, zi, and ai ionic fragments refer to the standard peptide fragmentation nomenclature: a, b, and c correspond to ionic fragments with the charge on the N terminal side while x, y, and z are for ionic fragments with the charge on the C terminal side.17,18 The ammonia loss occurs in [WG]H+ and [WL]H+ molecules when the chromophore is close to the N terminal side. It can be anticipated that the basic photofragmentation mechanisms resulting from the excitation of the indole chromophore have many common aspects with that of the protonated tryptophan investigated previously2,5 although new fragmentation channels, i.e., the peptide chain fragmentation, is present. To rationalize the important set of data (see Table 1), the WH+ fragmentation scheme will then be used as a guideline for the presentation of the results. Taking [GWG]H+ as a prototype (Figure 1b), fragmentation is triggered when the excited electron is localized on the chromophore (site 3) or transferred to orbitals localized on the amino group (site 1), on one of the carbonyl (sites 2, 4), or on the carboxyl (site 5). The proposed fragmentation pathways for each site are shown in Figure 4 and discussed below. III.1. Active Electron Attached to the N Terminal Site (Site 1): H Loss. The H loss from a m/z species leaving a m/z - 1 fragment ion is a very important photodissociation channel in WH+.12,19 With the present set up, detection of this fragment at the exit of the electrostatic analyzer is not possible because of the small mass difference with that of the incident ion, the corresponding signal being hidden in the dominant contribution of the incident beam. Therefore, this channel cannot be investigated in our experiment with great detail (coincidence measurements). As in WH+ fragmentation, the m/z - 1 radical cation might, however, undergo an additional delayed fragmentation (τ > 20 µs).5 In the WH+ case, this pathways results in the emission of a slow m/z 130 ion due to CR-Cβ breaking allowing possible indirect identification of the H loss channel.5,20 In these experiments the laser was fired in the trap. If formed, the long living m/z - 1 fragment ion is stored along with the parent ion as long as it does not dissociate. Such subsequent fragmentation results in the production of neutral fragments escaping the trap and reaching the neutral PSD. In the present case, for all investigated dipeptides, we do not find any significant delayed fragmentation of the m/z - 1 radical cation. This means that either there is no H loss or the fragmentation

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TABLE 1: Photofragmentations Channels for the Four Protonated Dipeptidesa NH3 loss WH+ m/z 205 [WG]H+ m/z 262 [WL] H+ m/z 318 [GW]H+ m/z 262 [LW]H+ m/z 318 [GWG]H+ m/z 319

a

ion fragment mass no. of steps fragment. time (ns) probability (%) ion fragment mass no. of steps fragment. time (ns) probability (%) ion fragment mass no. of steps fragment. time (ns) probability (%) ion fragment mass no. of steps fragment. time (ns) probability (%) ion fragment mass no. of steps fragment. time (ns) probability (%) ion fragment mass no. of steps fragment. time (ns) probability (%) +

188 1 10 13 245 1 20 8 301 1 110 53

side chain side chain CR-Cβ CR-Cβ z1 or z2 CO + H2O loss a1 or a2 y1 or y2 130 1 20 5 130 1 45 3 130 1 50 2 130 1 50 6

132 1 20 15 132 1 25 7 132 1 85 6 132 g2

130 1 150 2

132 2

11

5

159 2 20 10 159 2 30 48 159 2 130 34 188 1 70 18 188 2 >200 5 245 1 70 19

216 2 60 14

216 2 100 46

205 2 60 15 205 2 >200 90 262 2 50 21

delayed fragmentations 144-146 2 >1000 42 144 2 >1000 21 144 2 >1000 5 188 3 >1000 36 188 3 >1000 5

130 2 .1000 15 244 2 >1000 6

130 2 >1000 7

244 2 >1000 7

+

The results of WH and [GWG]H are recalled. For each fragmentation pathway are indicated the mass of the ionic fragment, the number of fragmentation steps, the fragmentation time, and the relative probability.

of the radical cation is too fast to decay in the trap (τ < 5 µs). Assuming such fast fragmentation, two decay pathways, as described below, can be examined (Figure 4). The ammonia loss has been observed as the main fragmentation channel in low energy CID of the WG+ radical cation.21 Indeed, in [WG]H+, we observe the m/z 244 ion fragment produced outside the polarized box a few microseconds after the laser shot, corresponding to a water loss or to a successive H and NH3 loss. However, the long fragmentation time in the microsecond regime implies that the observed loss is due to a secondary fragmentation that favors the interpretation in terms of successive H and NH3 losses. An other possible decay pathway involves slow CR-Cβ breaking as already observed in WH+5,20 giving m/z 130 fragments. Such slow formation of m/z 130 fragments is presently observed in [WG]H+ at ti - tn ) 1.7 µs (Figure 5a) as a secondary fragmentation pathway following the fast H loss. Notice that such slow fragmentations governed by H loss including slow CR-Cβ breaking is not observed in the other systems investigated. The decay of the radical cation left after the H loss is much faster in [WG]H+ than in WH+, suggesting that these dipeptides are warmer. III.2. Active Electron Attached to the N Terminal Site (Site 1): NH3 Loss and m/z 144 Production. Another pathway observed in the WH+ fragmentation corresponds to the single loss of ammonia.22 This channel is also found with small peptides, but only when the indole is close to the N terminal.23 This is presently the case for [WG]H+ and [WL]H+ ions (Table 1) for which the m/z 245 and m/z 301 fragments are respectively produced. This pathway is not observed with [GW]H+, [LW]H+, and [GWG]H+ species. The dissociation occurs in one step, as shown by the “VV” contour plots (not presented here). The fragmentation times are rather short, 20 and 110 ns for [WG]H+ and [WL]H+, respectively. Such fragmentation was previously interpreted as resulting from an IC mechanism.5 However, recent experiments and calculations on tryptophan complexed with

crown ether24,25 put such interpretation in question and suggests that this pathway involves an electron transfer from the indole chromophore to an antibonding orbital, the πσ* configuration (site 1), located between C and NH3. This intermediate breaks again to produce m/z 144 ion fragments (see Table 1). The corresponding N(Yi,ti - tn) diagram (Figure 6a) only shown for [WG]H+ indeed indicates a two-step fragmentation producing n1 and n2 neutral fragments associated with a unique m/z 144 ion fragment. The pattern at ti - tn ) 2.2 µs corresponds to the first fragmentation, a fast fragmentation occurring inside the interaction box. The second fragmentation is responsible for the pattern appearing at ti - tn < 1.5 µs. An analysis of the triple (i, n1, n2) coincidence events corresponding to this twostep fragmentation has been done. The “VV” contour plot between i and n2 (Figure 6b) allows us to determine the n2 mass (m2 )101) from which one deduces that of n1 (m1 ) 17). The slope of the “VV” contour plot between the n1 neutral fragment and the center of mass of the m/z 144 ion and n2 (Figure 6c) confirms this two-step mechanism, namely a fast ammonia loss followed by a slow fragmentation of m/z ) 245 (262 - 17), which then leads to the m/z 144 production (Figure 4). Note that such sequential fragmentation can easily be disentangled5 because the two fragmentation times are very different. III.3. Active Electron Attached to CO Close to the Chromophore on the C Terminal Side (Site 2 or 5): Cr-Cβ Bond Breaking. For the presently investigated molecules, the CR-Cβ bond breaking should result in the production of m/z 130 and m/z 132 fragments with H and H+ bound to the indole ring. Table 1 shows that these fragmentation pathways, found in WH+, are much less populated in protonated dipeptides. These channels are too weak in [LW]H+ to be studied. Figure 5 shows the N(Yi,ti - tn) contour plots for [WG]H+, [WL]H+, [GW]H+, and [GWG]H+ species. Depending on the molecule, the m/z 130 fragments can be produced along two different pathways, corresponding to fast and slow dissociations. Concerning the fast fragmentation,



Figure 4. Scheme of the various pathways. Pathways involving sites 1-3 are shown for [XW]H+ molecules and pathways involving sites 4-5 are shown for [WX]H+ molecules. X stands for the additional peptide with the R residue. The pathway producing m/z 188 by internal conversion is also shown.22,29

for all systems the m/z 130 “VV” contour plots (not shown here) reveal that this channel results from a pure binary fragmentation (Table 1). This mechanism discussed in refs 2, 6, and 26 involves a concerted transfer of the active electron on the π*CO (site 2 or 5), and of the proton on the carbonyl that results in a weakening of the CR-Cβ bond leading to the m/z 130 fragmentation. While the fast dissociation pathways are present in the four systems, the slow dissociation only occurs in [WG]H+(see section III.1).

III.4. Active Electron Stays on the Chromophore (Site 3): Cr-Cβ Bond Breaking with H+ and H Transfers. Table 1 shows that, for direct fast fragmentations, the m/z 132 is more populated than the m/z 130 for all systems including WH+.5 The “VV” contour plots for [WG]H+ and [GW]H+ for m/z 132 are shown in Figure 7. They show very different patterns that clearly demonstrate they involve different dissociation mechanisms: a binary fragmentation for [WG]H+ and [WL]H+ (not shown here) and a multistep fragmentation for [GW]H+

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Figure 5. N(Yi,ti - tn) contour plots for three dipeptides and a tripeptide. The data are obtained with the interaction box polarized at 25 V/mm (a) and (b), 16 V/mm (c), and 12 V/mm (d). Crosses: calculated positions of the m/z ions fragmented inside the interaction box (fast fragmentations). Stars: calculated positions of ions fragmented between the polarized box and the entrance of the electrostatic analyzer (slower fragmentations).

fragmentation and [GWG]H+ (not shown here). This indicates that binary fragmentation occurs in systems in which the indole chromophore is located close to the N terminal (Figure 2) making easy an H and H+ transfer on the indole ring. In [GW]H+ and [GWG]H+ the NH3 group is too far from the indole ring to allow the excited state proton transfer from the NH3 to the indole, which has been shown to be the first step of the m/z 132 formation in WH+.26 Notice that this topological effect occurs because of an entropic effect due to the rather high temperature of the molecule: indeed at very low temperature the strong interaction between the NH3+ and the π cloud of the indole ring locks the NH3 near the ring and thus favors the NH3-indole proton transfer. At high temperature, the peptide chain is completely unfolded and the indole ring is far from the ammonia for [GW]H+, [LW]H+, and [GWG]H+. The m/z 132 multistep fragmentations observed in this last case ([GW]H+ and [GWG]H+) cannot presently be explained. III.5. Active Electron Attached to CO on the N Terminal Side (Site 4): N-Cr Breaking Producing z1 or z2 Ions. The localization of the active electron on the CO of the N terminal side should trigger fragmentations that produce z1 ions for dipeptides and z1 and z2 ions for tripeptides. It is noteworthy that the z1 and z2 fragmentations, characteristic pathways in ECD,27 require first an electron attachment to the carbonyl followed by a proton transfer to the carbonyl group that weakens the N-CR bond.28 This pathway only occurs in [GW]H+, [LW]H+, and [GWG]H+ species. This is an important and fast channel for [GWG]H+ and [GW]H+ (Table 1) resulting from a binary fragmentation (Figure 8 and 10b). This channel, with a fragmentation time larger than 200 ns is much weaker in [LW]H+ (table 1) and cannot be fully analyzed.

III.6. Active Electron on the Carboxyl Group (Site 5): H2O and CO Losses. This situation was studied in detail for the photofragmentation of [W]H+ species. The mechanism involves a fast two-step fragmentation with a successive loss of H2O and CO.5 It is not surprising that the same process is found in [GW]H+ species, not shown here, since the carboxyl is close to the chromophore. It could have been anticipated that the same mechanism would also occur in [LW]H+. This is not the case; this pathway may perhaps be suppressed by steric hindrance. III.7. Internal Conversion: Cleavage of the Amide Bond Giving a1 and y1 Ion Fragments. The channels resulting from internal conversion are the most important pathways in low energy CID.18 Notice that they are also observed in PID.9,10 For dipeptides, the a1 cleavages occur in [WG]H+ and [WL]H+ (Figure 2) with the tryptophan located on the N terminal and the y1 cleavages occur in [GW]H+ and [LW]H+ with the tryptophan located on the C terminal sites. For [GWG]H+ tripeptide both a2 and y2 cleavages occur. Table 1 shows that these channels are also very important here for the investigated protonated dipeptides and the [GWG]H+ tripeptide. The corresponding “VV” contour plots are shown in Figure 9 (top row). A binary fragmentation would give a linear pattern aligned along the dotted line corresponding to the ratio between the mass of the ion fragment and that of the single neutral fragment. This is clearly not the case, thus we can conclude that these fragmentations occur through a multistep process. A first possible two-step fragmentation would consist of the ejection of the ion fragment first followed by a subsequent fragmentation of the remaining neutral into two fragments n1 + n2. This first scenario is eliminated since the corresponding “VV” contour plots would follow the dotted line (Figure 9) of



Figure 6. Two-step m/z 144 fragmentation pathway of [WG]H+: m/z 262 f m/z 245 + 17; m/z 245 f m/z 144 + 101. (a) N(Yi,ti - tn) contour plots for the m/z 144 fragment. The mass of the ionic fragments is identified by the position Yi on the MCP. The difference of arrival time ti - tn allows us to identify the ions fragmented inside the interaction box polarized at V ) 600 V.(RHS pattern) from that fragmented after they have left the box (LHS pattern). (b) “VV” contour plots corresponding to the correlation of the m/z 144 ion fragment and the neutral n2 fragment emitted in the second step outside the interaction box. The S ) 0.68 ( 0.03 slope of the pattern (dashed red line) given by a linear regression fit provides an estimate m ) 144 × 0.68 ) 98 ( 4 of the mass m ) 101 of n2. “VV” contour plots corresponding to the correlation of the m/z 144 ion fragment and the neutral n1 fragment ejected inside the interaction box. The 0.066 ( 0.03 slope of the pattern (dashed red line) given by a linear regression is in agreement with the (d) expected mass ratio (m17)/(m144+101) ) 0.069.

Figure 7. “VV” contour plots corresponding to the m/z 132 ion fragmentation in [WG]H+ and [GW]H+. The slope of the dashed lines is the ratio between the neutral mass fragment and the ionic mass fragment for a binary fragmentation. The slope s ) 1.0 of the [WG]H+ structure given by a linear regression compares well with the mneutral/mion ) 131/132 ) 0.984 mass ratio for [WG]H+.

the binary fragmentation with some additional broadening due to the neutral fragmentation. One can then investigate the other scenarios. For [WG]H+, [WL]H+, [LW]H+, and [GWG]H+ (m/z 216), the contour plot shows a superposition of two structures, a ball pattern superimposed to a cigar shape pattern, indicating that the two neutral fragments have very different masses. Conservation of linear momentum dictates that the lighter fragment will carry most of

Figure 8. “VV” contour plot showing the binary fragmentation of [GWG]H+ producing m/z 245 ion fragments. The slope of the dashed lines is the ratio between the neutral mass fragment and the ionic mass fragment for a binary fragmentation.

the kinetic energy. Selecting among the triple coincidence events, those between the most deviated neutral and the ionic fragment, the corresponding “VV” correlation exhibits a cigar structure stretched with a small slope corresponding to the ejection of the lighter neutral mass with a large KER (Figure 9 second row). Events inside the open rectangles (Figure 9 first raw), on the tips of the cigars, correspond to fragmentation

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Figure 9. Sequential peptidic bound fragmentation of [WG]H+, [WL]H+, [GW]H+, [LW]+, and [GWG]H+ First row: “VV” contour plots corresponding to the correlation between the ion and one neutral fragment, either n1 or n2. The slope (M0 - mi)/mi of the dashed line is given by the ratio between M0 - mi, the mass of the neutral fragment, and mi, that of the ionic fragment for a binary fragmentation. Second row: Triple coincident events. “VV” contour plots corresponding to the correlation between the ionic fragment and the most deviated neutral fragment, the one with the largest speed in the center of mass frame. The slope of the dotted line is given by a fit of the structure (see Table 2). The corresponding diagrams for [GW]H+ and [GWG]H+ are not shown because it is impossible to select the closely lying mn1 ) 28 and mn1 ) 29 masses of the two neutrals in triple coincident events. Third row: Triple coincident events. “VV” contour plots corresponding to the ionic fragment and the less scattered neutral fragment. For this purpose events inside the rectangles are selected. Notice that the poor statistic is due to the small number of events selected that way. The slope of the dotted line is given by a fit of the structure.

TABLE 2: Masses of the Two Neutral Fragments Assuming a Sequential Peptidic Bound Fragmentation of [WG]H+, [WL]H+, [LW]H+, and [GWG]H+ Depending Whether the Lighter Neutral Fragment Is Ejected in the First or Second Step peptide mass m/z ion fragment slope S

[WG]H+

[WL]H+

[LW]H+

[GWG]H+

262 159 0.12

318 159 0.103

318 205 0.085

319 216 0.098

N1 fragment N2 fragment

Light Neutral Fragment Ejected First 30 30 25 73 129 88

29 74

N1 fragment N2 fragment

Heavy Neutral Fragment Ejected First 84 143 96 19 16 17

82 21

producing two neutrals with very different KER. Selecting triple coincidence events inside these data, allows drawing the “VV” correlation between the ion fragment and the heavier neutral (Figure 9 third row), providing the mass of this slow neutral. The much larger slope clearly confirms the additional ejection of a heavier neutral fragment. Two scenarios are still possible for the two-step fragmentation depending whether n1 or n2 be ejected first. Unfortunately, the fragmentation steps are too fast to permit the selection of the fragmentation sequence as it is the case in section III.2. Therefore, one has to examine what are the two possible couples of neutral masses, depending whether the light fragment is emitted first or in the second step. The mass of the lighter neutral is then given by MnL ) SM/(1 + S) if it is ejected in the first step, and MnL ) SMI if it is ejected in the second step. M is the total mass, MI is that of the ion fragment, and S is the slope given by the cigar shape pattern

(Figure 9 second row). Table 2 shows the masses of the two neutral fragments given by both hypotheses. The first hypothesis leads, for the investigated molecules, to the ejection of a mass of about m ) 29 ( 2 as a first neutral that would correspond within the experimental uncertainty to a CO molecule ejected first. This hypothesis corresponds to a1 and y1 cleavages that are major CID pathways at low collision energy. They have been the subjects of several theoretical studies showing that such fragmentation involves a proton transfer from the N terminal to the amide group that requires 2 eV. The twostep fragmentation pathways that are found here in all cases are compatible with the Paizs and Suhai model29 that involves the formation of a proton bound complex with ejection of a CO neutral fragment in a first step. Harrison et al.18 suggested that the fragmentation involves an initial formation of a proton bound complex of an aziridinone and an amino -acid. Harrison et al.’s model predicts a two-step fragmentation for [WG]H+ and [WL]H+ but with emission of CO in second step and fails to explain a similar two-step fragmentation pathway for [GW]H+ and [LW]H+ which is predicted to be a binary fragmentation. The second hypothesis assumes the ejection of the light neutral in the second step. This would lead for the investigated molecules, to the ejection of a neutral fragment with a mass m ) 19 ( 2, that could be a water or ammonia molecule. However, from energetic considerations coming from theory, such a fragmentation does not seem to be possible.29,30 For [GW]H+ and [GWG]H+ (m/z 262), the patterns are consistent with the emission of two neutral fragments of similar masses, a situation that is difficult to unravel, but most probably the processes are similar; i.e., two neutrals are emitted with CO lost as the first step.



Figure 10. (a) N(Yi,ti - tn) contour plot for the m/z 188 fragmentation of [GW]H+ of mass M0 ) 262. The R structure located at ion energy Ei ) 3300 eV corresponds to a fast binary fragmentation occurring in the box polarized at V ) 1000 V. In this case: mion_f Ei ) (EO + V) -V M0 The two structures at Ei ) 3390 eV correspond to the same physical event with a first fragmentation occurring inside the polarized box (β structure) leading to the formation of an ion of mass mion_int. Then the second fragmentation occurs outside the box leading to a final fragment ion of mass mion_f ) 188. From the final ion energy mion_f mion_f Ei ) (EO + V) -V ) 3390 eV M0 mion_int we deduce mion_int ) 205. (b) “VV” contour plot of the R events. The slope of the dashed lines is the ratio between the neutral fragment of mass 74 and the ionic fragment of mass 188 corresponding to a binary fragmentation. (c) “VV” contour plot of the β events inside the box. The slope of the dashed lines is the ratio between the neutral fragment of mass 57 and the intermediate ionic fragment of mass 205 that would correspond to a binary fragmentation. The disagreement between the two slopes clearly shows that the fragmentation inside the box is not binary.

In the [GWG]H+ tripeptide, the mobile proton can migrate to two different amide sites giving the y2 and a2 cleavages and producing m/z 262 and m/z 216, respectively. The a2 fragmentation is favored with respect to y2 (Table 1). III.8. Additional Fragmentation of the y1 Fragment in [GW]H+ and [LW]H+. The y1 fragment issued from in [GW]H+ and [LW]H+ is m/z 205 fragment (protonated tryptophan). The fragmentation channel of [W]H+, which appends at the lowest energy is the loss of NH3.22 We will see that this fragmentation channel occurs also in [W]H+ obtained from [GW]H+ after the loss of two neutral fragments. The N(Yi,ti - tn) contour plot (Figure 10) for the fragmentation of [GW]H+ species shows an important contribution located near yi ) 5 mm (Figure 10a) in addition to the m/z 188 fragmentation at yi ) -11 mm and discussed in section III.5. The top contribution appears as two structures corresponding to fragmentation inside (ti - tn ) 3.6 µs) and outside (ti - tn ) 1.6 µs) the box. These two structures located at the same energy of 3390 eV correspond to a new sequential fragmentation

pathway producing m/z 188 ionic fragments. From the energy measurement of the final ion fragment m/z 188 of 3390 V, we can deduce that the first step inside the box leads to an intermediate ion fragment of m/z 205. A binary fragmentation would correspond to an emission of a neutral fragment of mass 57. This would give a “VV” contour map of slope S ) 57/205 ) 0.27 (Figure 10 c). The “VV” contour plot, corresponding to the dominant β structure, shows that the first step results from a fast sequential fragmentation (τ e 60 ns). This first step fragmentation produces y1 ion fragment, with CO and m ) 29 neutral losses as discussed above. The production of the final m/z 188 ion fragments involves an additional delayed (τ > 1 µs) ammonia loss from the intermediate m/z 205 ion fragment. The same multiple dissociation resulting in the production of three neutral fragments is also observed in [LW]H+ species. However, this process is much weaker in [LW]H+ and the fragmentation time leading to y1 (m/z 205) ions is much longer (see Table 1) than for [GW]H+. This result is not surprising because in [LW]H+, the larger degrees of freedom of leucine

3156


as compared to glycine, implies that more internal energy is deposited in the leucine fragment than in the glycine fragment. Thus the y1 fragment has smaller internal energy when coming from [LW]H+ and therefore less fragmentation. III.9. Fragmentation Times. It has been shown in previous papers on photofragmentation produced by femtosecond laser pulses9,12,19,31 that the excited states of protonated aromatic amino acids decay in times shorter than a few picoseconds. In tryptophan, it has been shown that the very short lifetime of the excited state is governed by the hydrogen dynamics in the excited states, which trigger the excited state dissociation.6 The present experiments are done in a much longer time scale from nanoseconds to milliseconds in which new dynamics can be expected. In the nanosecond time scale, the present studies show that the fragmentation times scale with the size of the molecule, from about 10 ns for [W]H+, 20 ns for [GW]H+ and [WG]H+ to 100 ns for [LW]H+, [WL]H+, and [GWG]H+. These lifetimes are very long for excited state dynamics since they should be smaller than the fluorescence lifetime, which is smaller than 20 ns. The present lifetime increasing with the size of the system suggests a statistical behavior. Thus our interpretation is the following: the polarization interaction between the ionic and neutral fragment is not small (typically 1 eV) and the dissociated system, in the chemical sense, is trapped in this ion-neutral complex and fragments through a statistical process, a mechanism that can take a long time after some energy has been lost to break the chemical bond (2 or 3 eV). This readily explains that the fragmentation time scales with the molecular size. Consider now fragmentations occurring after internal conversion (IC) in the hundreds of femtosecond time scale, as shown by femtosecond experiments.12 The internal energy is the sum of the photon energy and of the vibrational energy due to the initial temperature. This internal energy has to be compared to the reaction barrier, which for example, is about 2.5 eV in the Paizs and Suhai model.29 The net result is that the fragmentation time is very similar to the ones triggered by excited state dynamics. This model also explains the nice results of Guidi et al.1 In their experiments, the aromatic chromophore of a protonated peptide was excited by a UV laser. The molecules were then re-excited by a CO2 laser, increasing their vibrational energy and hence their dissociation rate, allowing detection of the UV excitation by monitoring the photofragment intensity. The low initial temperature and the large size of the molecules prevent the excited state fragmentation from occurring unless some internal energy is brought into the system by the CO2 laser. In our experiment, the molecules are smaller and the temperature higher; therefore, the fragmentation occurs much more rapidly. Dissociation occurring at a longer time scale (>100 ns) should result from secondary fragmentations. It can hardly be thought that direct fragmentation could be very slow. Indeed, a multiplestep fragmentation is clearly revealed by the “VV” correlations in which the ion parent is clearly identified, as for the m/z 144 produced in the [WG]H+ and [WL]H+ fragmentations. IV. Conclusion In the present paper we have disentangled the photofragmentation mechanisms of small peptide containing a tryptophan residue. The general picture used for the description of the photofragmentation of protonated tryptophan has been extended to these larger systems. Two types of fragmentation are evidenced: some fragmentations are triggered by the hydrogen transfer in the excited states followed by a C-C or C-N bond rupture, but the fragments are trapped in the exit channel by the ion-molecule interaction. The other channels result from vibrationally induced fragmentation of the ground state reached via internal conversions, thus a

Pe´rot et al. fragmentation very similar to the one observed in CID. In this case our experiment allows following the dynamics in real time of such a process and gives the ordering of the multistep fragmentation even when the two steps occur within a few nanoseconds. In this experiment we have shown that two fully different mechanisms can give the same m/z fragment, information that cannot be easily obtained in usual mass spectrometry experiments. Acknowledgment. The present work has been carried out at the “Centre de Cine´tique Rapide ELYSE”. The CNRS and the “Universite´ Paris-Sud” are thanked for their financial support. References and Notes (1) Guidi, M.; Lorenz, U. J.; Papadopoulos, G.; Boyarkin, O. V.; Rizzo, T. R. J. Phys. Chem. A 2009, 113, 797. (2) Lucas, B.; Barat, M.; Fayeton, J. A.; Perot, M.; Jouvet, C.; Gregoire, G.; Nielsen, S. B. J. Chem. Phys. 2008, 128, 7. (3) Reilly, J. P. Mass Spectrom. ReV. 2009, 28, 425. (4) Joly, L.; Antoine, R.; Broyer, M.; Dugourd, P.; Lemoine, J. J. Mass Spectrom. 2007, 42, 818. (5) Lepe`re, V.; Lucas, B.; Barat, B.; Fayeton, J. A.; Picard, Y.; Jouvet, C.; Çarçabal, P.; Nielsen, I.; Dedonder-Lardeux, C.; Gre´goire, G.; Fujii, A. J. Chem. Phys. 2007, 127, 134313. (6) Gregoire, G.; Lucas, B.; Barat, M.; Fayeton, J. A.; DedonderLardeux, C.; Jouvet, C. Eur. Phys. J. D 2009, 51, 109. (7) Stearns, J. A.; Mercier, M.; Seaiby, C.; Guidi, M.; Boyarkin, O. V.; Rizzo, T. R. J. Am. Chem. Soc. 2007, 129, 11814. (8) Gregoire, G.; Jouvet, C.; Dedonder, C.; Sobolewski, A. L. J. Am. Chem. Soc. 2007, 129, 6223. (9) Nolting, D.; Schultz, T.; Hertel, I. V.; Weinkauf, R. Phys. Chem. Chem. Phys. 2006, 8, 5247. (10) Gregoire, G.; Kang, H.; Dedonder-Lardeux, C.; Jouvet, C.; Desfrancois, C.; Onidas, D.; Lepere, V.; Fayeton, J. A. Phys. Chem. Chem. Phys. 2006, 8, 122. (11) Gregoire, G.; Dedonder-Lardeux, C.; Jouvet, C.; Desfrancois, C.; Fayeton, J. A. Phys. Chem. Chem. Phys. 2007, 9, 78. (12) Kang, H.; Dedonder-Lardeux, C.; Jouvet, C.; Gregoire, G.; Desfrancois, C.; Schermann, J. P.; Barat, M.; Fayeton, J. A. J. Phys. Chem. A 2005, 109, 2417. (13) Lucas, B.; Barat, M.; Fayeton, J. A.; Jouvet, C.; Carcabal, P.; Gregoire, G. Chem. Phys. 2008, 347, 324. (14) Pedersen, H. B.; Strasser, D.; Ring, S.; Heber, O.; Rappaport, M. L.; Rudich, I.; Sagi, I.; Zajfman., D. Phys. ReV. Lett. 2001, 87, 55001. (15) Pedersen, H. B.; Stasser, D.; Heber, O.; Rappaport, M. L.; Zajfman, D. Phys.ReV. A 2002, 65, 042703. (16) Pedersen, H. B.; Stasser, D.; Amarant, B.; Heber, O.; Rappaport, M. L.; Zajfman, D. Phys. ReV. A 2002, 65, 042704. (17) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601. (18) Harrison, A. G.; Csizmadia, I. G.; Tang, T. H.; Tu, Y. P. J. Mass Spectrom. 2000, 35, 683. (19) Kang, H.; Dedonder-Lardeux, C.; Jouvet, C.; Martrenchard, S.; Gregoire, G.; Desfrancois, C.; Schermann, J. P.; Barat, M.; Fayeton, J. A. Phys. Chem. Chem. Phys. 2004, 6, 2628. (20) Talbot, F. O.; Tabarin, T.; Antoine, R.; Broyer, M.; Dugourd, P. J. Chem. Phys. 2005, 122. (21) Bagheri-Majdi, E.; Ke, Y. Y.; Orlova, G.; Chu, I. K.; Hopkinson, A. C.; Siu, K. W. M. J. Phys. Chem. B 2004, 108, 11170. (22) Lioe, H.; O’Hair, R. A. J.; Reid, G. E. J. Am. Soc. Mass Spectrom. 2004, 15, 65. (23) Poulain, P. http://tel.archives-ouvertes.fr/docs/00/09/47/47/PDF/ these-poulain, 2006. (24) Kadhane, U.; Andersen, J. U.; Ehlerding, A.; Hvelplund, P.; Kirketerp, M. B. S.; Lykkegaard, M. K.; Nielsen, S. B.; Panja, S.; Wyer, J. A.; Zettergren, H. J. Chem. Phys. 2008, 129, 5. (25) Kadhane, U.; Perot, M.; Lucas, B.; Barat, M.; Fayeton, J. A.; Jouvet, C.; Ehlerding, A.; Kirketerp, M. B. S.; Nielsen, S. B.; Wyer, J. A.; Zettergren, H. Chem. Phys. Lett. 2009, 480, 57. (26) Gregoire, G.; Jouvet, C.; Dedonder, C.; Sobolewski, A. L. Chem. Phys. 2006, 324, 398. (27) Zubarev, R. A. Mass Spectrom. ReV. 2003, 22, 57. (28) Syrstad, E. A.; Turecek, F. J. Am. Soc. Mass Spectrom. 2005, 16, 208. (29) Paizs, B.; Suhai, S. Rapid Commun. Mass Spectrom. 2001, 15, 651. (30) Paizs, B.; Suhai, S. Mass Spectrom. ReV. 2005, 24, 508. (31) Kang, H.; Jouvet, C.; Dedonder-Lardeux, C.; Martrenchard, S.; Gregoire, G.; Desfrancois, C.; Schermann, J. P.; Barat, M.; Fayeton, J. A. Phys. Chem. Chem. Phys. 2005, 7, 394.

JP908937S

Mechanisms of UV Photodissociation of Small Protonated Peptides

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