UV Photodissociation of Proline-containing Peptide Ions: Insights from Molecular Dynamics
UV photodissociation of proline-containing peptide ions leads to unusual product ions. In this paper, we report laser-induced dissociation of a series of proline-containing peptides at 213 nm. We observe specific fragmentation pathways corresponding to the formation of (y-2), (a + 2) and (b + 2) fragment ions. This was not observed at 266 nm or for peptides which do not contain proline residues. In order to obtain insights into the fragmentation dynamics at 213 nm, a small peptide (RPK for arginine-proline-lysine) was studied both theoretically and experimentally. Calculations of absorption spectra and non-adiabatic molecular dynamics (MD) were made. Second and third excited singlet states, S2 and S3, lie close to 213 nm. Non-adiabatic MD simulation starting from S2 and S3 shows that these transitions are followed by C-C and C-N bond activation close to the proline residue. After this first relaxation step, consecutive rearrangements and proton transfers are required to produce unusual (y-2), (a + 2) and (b + 2) fragment ions. These fragmentation mechanisms were confirmed by H/D exchange experiments.
Key wordsPhotodissociation Proline-containing peptides Fragmentation Molecular dynamics H/D exchange
Tandem mass spectrometry (MS/MS) is a widely-used method to determine the amino acid sequence of peptides and proteins . In addition, the use of CID (collision-induced dissociation) continues to be important in peptide sequencing and protein identification. In solution, the most basic site on a peptide devoid of histidine, lysine, and arginine residues is the nitrogen atom of the N-terminal amino group . However, once the protonated peptide ion is desorbed into the gas phase, e.g. via electrospray, there is competitive transfer of the “ionizing” proton to amidic functional groups, the carbonyl oxygen and amidic nitrogen atoms on the backbone [3, 4, 5, 6, 7, 8]. The peptide then fragments at the protonated peptide bond [9, 10]. A non-terminal residue has two adjacent peptide linkages, one to the N-terminal and the other to the C-terminal. At low (<100 eV) energy CID, the protonated peptides fragment along the backbone at the amide bonds, and the mass spectrum typically consists of a series of b, a, and y fragment ions. These fragments give valuable information [9, 10] concerning peptide sequence, number and location of disulfide bridges, identification and sometimes characterization of post-translational modifications. However, fragmentation pathways of protonated peptides are difficult to understand and known only at the phenomenological level.
It is apparent from the diverse CID patterns observed that the fragmentation routes of protonated peptides depend significantly on the identity and positions of the amino acids constituting the peptides. It was found that proline (Pro) and to a smaller extent glycine and serine residues tend to fragment at their N-terminal peptide bonds, whereas residues such as valine and isoleucine tend to fragment toward their C-terminal peptide bonds . Other published work using tandem MS on peptides, e.g., ubiquitin, have often indicated unusually abundant product ions resulting from cleavage of peptide linkages on the N-terminal side of Pro residues . This propensity for selective fragmentation has also been reported for singly-protonated peptides [13, 14, 15, 16, 17, 18] and multiply-charged protein ions . This phenomenon is sometimes known as the proline effect and was first attributed to the relatively high proton affinity of Pro . Vaisar and Urban investigated a number of peptides that contained a modified amino acid residue of relatively high proton affinity, most notably one that contained a six-membered piperidine ring as opposed to Pro’s five-membered pyrolidine ring, and concluded that proton affinity does not explain the proline effect . They attributed the preferred N-terminal (as opposed to C-terminal) cleavage to Pro in terms of increased ring strain in forming a bicyclic b ion when the C-terminal peptide bond is cleaved. The conformation of the Pro residue may also play an important role in fragmentation. Wysocki and co-workers studied this with a database of low-energy CID tandem mass spectra of doubly-charged peptides . They found that the residue (Xxx) adjacent to Pro affects the extent of fragmentation at the Xxx-Pro bond. Particularly enhanced Xxx-Pro bond cleavage when Xxx is Val, Ile, and Leu was ascribed to the steric hindrance of their bulky side chains, since it causes Pro to take a ‘reactive’ trans form that leads to product ions. However, they did not elucidate the ‘reactive’ conformation of Pro or the fragmentation mechanism. Infrared multiphoton dissociation (IRMPD) of peptides results in few backbone (b- and y-type) cleavages but increased side-chain fragmentation. In addition, IRMPD showed an increased selectivity toward N-terminal backbone cleavages to Pro .
In addition and complementary to CID, reactions of polypeptide ions with electrons and small radical ions, such as electron capture dissociation (ECD) [23, 24] and electron-transfer dissociation (ETD) [25, 26, 27], have become very useful tools for peptide structural analysis. The ion–electron and ion–ion reactions are different from slow heating methods, such as CID, by the fact that the intermediate fragmenting species are odd-electron ions. In fact, the presence of a radical site diminishes the strength of nearby bonds. In particular, for peptide polyanions, preferential backbone cleavage of C–C bonds yielding a and x ions as well as side chain fragmentation were demonstrated [28, 29, 30, 31, 32]. Specific cleavage of peptide linkages on the N-terminal of Pro residues was also observed in ECD [30, 33, 34].
Vacuum ultraviolet photodissociation (VUVPD) is an elegant, high-energy method for inducing fragmentation in peptides . Following electronic transition, direct dissociation in the excited state competes with radiative relaxation and internal conversion to the electronic ground state. VUVPD of Pro-containing peptide ions was reported with the production of unusual product ions . In particular, unusual bn + 2 and an + 2 ions were observed. Their formation was explained by homolytic cleavage of the Cα-C bond in conjunction with a rearrangement between electrons and an amide hydrogen. Formation of these abnormal ions has been compared to the effect of Pro on gas-phase conformation of peptides. UVPD of peptides at 193 nm was achieved with production of a, b, c, x, y, and z sequence ions, in addition to immonium ions and v and w side-chain loss ions . However, the unusual fragment ions were not reported for the studied sequences.
Herein, we investigate the UVPD of Pro-containing peptides at 213 nm. This wavelength corresponds to the emergence of the lowest-lying electronic excited states of the molecules and thus allows the investigation of the relation between the nature of excited states and the observed fragmentation patterns. The calculation of the electronic excited states and molecular dynamics for the model RPK peptide (arginine-proline-lysine) are reported here in order to gain insight into the first steps of UV photodissociation of Pro-containing peptides. Our complementary theoretical and experimental investigations open a new route for identification of the photofragmentation pathways allowing identification of the link between the nature of electronic excited states and the observed fragmentation pathways.
2 Material and Methods
Methanol (MeOH) was obtained from Fisher Scientific (Strasbourg, France) and milli-Q water (18.2 MΩ.cm) was used. Deuterated methanol (CH3OD), deuterium oxide (D2O), Bradykinin and Substance P acetate salts were purchased from Sigma–Aldrich (St Quentin-Fallavier, France). LGPLVEQGR, LGADMEDVR and EANYIGSDK peptides were synthesized by Millegen (Labège, France). RPK peptide was synthesized by GeneCust (Dudelange, Luxembourg).
2.3 Mass Spectrometry Operating Conditions
Ionization was achieved using an electrospray in positive ionization mode with an ion spray voltage of 4 500 V. The sheath gas and auxiliary gas (nitrogen) flow rates were set at 20 and 15 (arbitrary unit), respectively, with a HESI vaporizer temperature of 250°C. The ion transfer capillary temperature was 250°C. The S-lens RF was set at 90 (arbitrary unit). The orbitrap resolution was 140 000. The Automatic Gain Control (AGC) target was 3e6 and the maximum injection time was set at 250 ms. For photodissociation experiments, the HCD parameters were optimized in order to avoid CID and were set at 2 eV for the collision energy and 1000 ms for the activation time. CID experiments were performed using a normalized collision energy of 25% and 3 ms activation time. An m/z window of 2.0 Th was applied for precursor isolation. Peptides were dissolved in 50/50 MeOH/water (vol/vol) at a concentration of 100 μM and directly electrosprayed at a flow rate of 5 μL/min.
Investigation of the structures of the RPK model system (arginine + proline + lysine) was done using the simulated annealing method coupled to molecular dynamic simulations using the semi-empirical AM1 method . The structures found were then re-optimized using the semi-empirical OM2 (orthogonalization model 2) method . To calculate the absorption spectra, the OM2 method combined with the graphical unitary group approach (GUGA) multi-reference configuration interaction (MR-CI) [40, 41] within the MNDO program  was used. The active space which consists of five occupied and five virtual orbitals was chosen, and all single, double, and triple excitations out of the self-consistent field reference configurations were included in the calculations.
Since the experiments were done at a temperature close to 300 K, we simulated the thermally-broadened absorption spectra. The configurations were sampled from a long molecular dynamic trajectory run at a constant temperature of 300 K using the semi-empirical OM2 method. To simulate the temperature broadening of the spectrum, the absorption spectrum for each configuration was calculated and these spectra were superimposed. The non-adiabatic dynamics “on the fly” in electronic excited states were determined using Tully’s surface-hopping algorithm with non-adiabatic couplings . The initial conditions for non-adiabatic dynamics were obtained by sampling 100 coordinates and momenta along a 30 ps ground-state trajectory at a constant temperature of 300 K using the OM2 method. The nuclear trajectories were propagated by numerical solution of Newton’s equations of motion using the Verlet velocity algorithm  with a time step of 0.1 fs. The non-adiabatic dynamics were determined starting from S2 or S3 state matching experimental conditions.
3 Results and Discussion
3.1 The Photodissociation of Pro-containing Peptides
Exact masses and assignments of fragment ions detected in the photodissociation and CID spectra of doubly-protonated Bradykinin [M + 2H]2+ (Figure 2). Error = (m/z theoretical – m/z experimental)/(m/z theoretical)
[M + H]+
[M + H-C3H7O]+
(b6 + 2)+
(a6 + 2)+
[M + 2H]2+
[M + 2H-H2O]2+
(b2 + 2)+
(a2 + 2)+
(b1 + 2)+
(a1 + 2)+
More interestingly, photodissociation at 213 nm generates new fragments that are not observed in the CID spectrum. The assignment of these ions was confirmed by the exact masses (Table 1). Fragment ions detected at m/z 902.46, m/z 805.40 and m/z 417.22 correspond to the elemental composition of y ions minus 2 hydrogens and have been labeled in Figure 2a (y8-2)+, (y7-2)+ and (y3-2)+, respectively. These yn-2 ions are not observed along the whole peptide sequence. This fragmentation pathway occurs only with a Pro residue. In fact, Bradykinin contains 3 Pro residues Pro2, Pro3 and Pro7 among a total of 9 amino acid residues giving rise to (y8-2)+, (y7-2)+ and (y3-2)+ ions. These ions are more intense compared to their homologue yn ions. The relative intensities of (y8-2)+, (y7-2)+ and (y3-2)+ ions correspond to 50%, 150% and 300% of the relative intensities of (y8)+, (y7)+ and (y3)+, respectively. Fragment ions detected at m/z 159.12, m/z 256.18 and m/z 644.35 have the elemental composition of b ions plus 2 hydrogens and, in Figure 2a, have been labeled (b1 + 2)+, (b2 + 2)+ and (b6 + 2)+, respectively. This fragmentation reaction is very efficient compared to the classical mechanism as the relative intensities of (b1 + 2)+, (b2 + 2)+ and (b6 + 2)+ ions represent 400, 500 and 700% of the relative intensities of their bn homologues. The same behavior is observed for the fragment ions detected at m/z 131.13, m/z 228.18 and m/z 616.36 which correspond to an ions plus 2 hydrogens and, in Figure 2a, have been labeled (a1 + 2)+, (a2 + 2)+ and (a6 + 2)+, respectively. Furthermore, these ions are only observed in the neighborhood of the Pro residues. The (×3)+ ion is detected at m/z 348.17 in the photodissociation spectrum. Kim et al.  also observed these unusual fragment ions in the UVPD of singly-charged Pro-containing peptides at 157 nm.
The same photodissociation experiment was done for the doubly-protonated [M + 2H]2+ of Substance P (RPKPQQFFGLM) (m/z 674.86) and LGPLVEQGR (m/z 484.77) peptides. The spectra are shown in Figures S1 and S2 in the Supporting material. Besides the backbone fragments yn, bn and an observed in photodissociation and CID, UVPD spectra show yn-2, bn + 2 and an + 2 fragment ions. For the doubly-protonated Substance P peptide which contains 2 Pro residues Pro2 and Pro4, (y10-2)+ and (y8-2)+ ions are detected at m/z 1190.60 and m/z 965.45 corresponding to y ions minus 2 hydrogens (according to exact masses). b and a fragment ions plus 2 hydrogens (a1 + 2)+, (b1 + 2)+, (a3 + 2)+ and (b3 + 2)+ are detected at m/z 131.12, m/z 159.12, m/z 356.19 and m/z 384.26, respectively. The (×7)+ ion is detected at m/z 640.28 in the photodissociation spectrum. All assignment errors were less than 1.3 ppm. In the photodissociation spectrum of doubly-protonated LGPLVEQGR, which has a Pro3 residue among a total of 9 amino acid (AA) residues, (y7-2)+, (a2 + 2)+ and (b2 + 2)+ are detected at m/z 796.43, m/z 173.13 and m/z 145.13, respectively (Figure S2). From these 3 examples, we can conclude that (yn-2)+ ions are only observed in photodissociation for n = (total number of AA - #Pro +1). These ions still contain the Pro residue. On the other hand, (bn + 2)+ and (an + 2)+ ions are only observed in photodissociation for n = (#Pro - 1), and they do not contain the Pro residue. These fragmentation pathways are observed only with cleavage of bonds between the amino acid N-terminal to Pro and the Pro residue. In order to confirm the contribution of the Pro residue in these fragmentation pathways, photodissociation experiments were done at 213 nm for doubly-protonated ions of LGADMEDVR (m/z 503.24) and EANYIGSDK (m/z 498.74) peptides that do not contain Pro. The UVPD spectra are presented in Figures S3 and S4, respectively. In these cases, photodissociation and CID spectra are similar. No (yn-2)+, (bn + 2)+ or (an + 2)+ ions are detected in photodissociation. Therefore, the effect of the excited Pro residue is clearly evident in these new mechanisms. Note that photodissociation experiments were also done at 266 nm for the Pro-containing peptides and new fragment ions were not observed (data not shown). The fact that no unusual fragments close to the Pro were observed means that a higher energy UV excitation (below 266 nm) is required to induce these specific photo-fragments.
3.2 Photodissociation and Optical Properties of the Protonated RPK Model Peptide
Exact masses and assignments of fragment ions detected in the photodissociation and CID spectra of protonated RPK [M + H]+ (Figure 3). Error = (m/z theoretical – m/z experimental)/(m/z theoretical)
[M + H]+
[M + H-H2O]+
[M + H-2H2O]+
[M + H-C3H7O]+
(b1 + 2)+
(a1 + 2)+
Since the experiment is performed at a temperature close to 300 K, we calculated the thermally-broadened spectrum at 300 K, shown in Figure 4b, and found that the highest density of transitions occurred around 213 nm due to S2 and S3 excited states. Therefore, in order to follow fragmentation pathways, the non-adiabatic dynamics were studied starting from the S2 and S3 states. However, as they both proceed through similar pathways, the equivalent fragmentation pathways were observed. Therefore, we present results obtained starting from the S2 state only.
3.3 Non-adiabatic Molecular Dynamics for the Fragmentation of Protonated RPK Peptide
3.4 Fragmentation Mechanisms of the Protonated RPK Peptide
The (b2 + 2)+ fragment ion, detected at m/z 159.12, would be formed when the Arg side chain bears the charge. The mechanism involves first C-C bond activation then breaking in the Pro residue (shown in Figure 5b) and finally, proton transfer from the amine of the lysine (Lys) residue to the carbonyl group of the Arg, according to Scheme 1b. The (×1)+ ion at m/z 173.06 is generated with the same mechanism if the charge is located on the Lys residue rather than the N-terminus.
In the same way, trajectories reported in Figure 5c lead to the Cα-C bond breaking on the N-terminal side of Pro which is a prerequisite for generating (a + 2) type fragments. Additional proton transfer from the CH2 of the Pro ring to the alkyl group of the Arg and elimination of a CO molecule (presented in Scheme 1c) would produce the (a1 + 2)+ ion detected at m/z 131.13 when the Arg side chain bears the charge. This fragmentation pathway would also lead to the (y2-2)+ fragment ion if the charge is located on the Lys residue at the time of the dissociation.
Exact masses and assignments of fragment ions detected in the photodissociation spectrum of singly-charged deuterated RPK [M + D]+ (Figure 6). Error = (m/z theoretical – m/z experimental)/(m/z theoretical)
[M + D]+
[M + D-D2O]+
[M + D-2D2O]+
d8-(b1 + 2)+
d7-(a1 + 2)+
Finally, the (a1 + 2)+ fragment is detected with a 7 Da mass increase (Figure 6) which is inconsistent with a proton transfer from the Lys amine as proposed by Kim et al.  which would have led to an 8 Da mass increase. Alternatively, a proton transfer from a CH2 group of the Pro ring, as proposed in Scheme 1c, would explain the observed 7 Da mass increase, i.e. five deuterium on the Arg side chain bearing the charge and two deuterium at the N-terminal. Moreover, we can note that the relative intensity between the (a1-2)+ and (a1)+ ions is the same (73%) before and after H/D exchange (Figures 3a and 6). This confirms the proposed mechanism where D transfer is not required. In fact, hydrogen migration is favored over deuterium transfer.
To conclude, we observed the formation of unusual (y-2), (a + 2) and (b + 2) fragment ions upon photodissociation of proline-containing peptides at 213 nm. The formation of these ions was not observed at 266 nm, or for non-proline-containing peptides. The RPK peptide was the smallest peptide for which we were able to observe these fragmentation pathways experimentally. Calculation of the electronic excited states for this peptide showed that S2 and S3 states could be excited at the experimental wavelength. Non-adiabatic molecular dynamic simulation (MD) starting from S2 and S3 excited states showed that this excitation was followed by C-C and C-N bond activation close to the proline residue. The MD revealed early relaxation mechanisms leading to the observed fragmentation pathways.
Our complementary theoretical and experimental investigations open new routes for identification of photofragmentation pathways. They enable the identification of the link between the nature of electronically-excited states and the observed fragmentation pathways. The MD simulations on the fly provide the foundation for a molecular understanding of the photochemistry of peptides under UV excitation. These trajectories revealed specific breaking of C-C and C-N bonds close to the proline residue. Consecutive rearrangements and proton transfers are required to produce the above unusual fragment ions and the fragmentation mechanisms were confirmed by H/D exchange experiments.
V.B.-K. and P. D. would like to thank the CNRS NCBA international laboratory. V.B.-K. gratefully acknowledges support from the Deutsche Forschungsgemeinschaft (DFG FOR1282) and Split-Dalmatia County. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013 Grant agreement N°320659).
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