Article pubs.acs.org/JPCA
Isotope Labeling Study of Retinal Chromophore Fragmentation Lihi Musbat,† Maria Nihamkin,† Shany Ytzhak,† Amiram Hirshfeld,‡ Noga Friedman,‡ Jonathan M. Dilger,§ Mordechai Sheves,‡ and Yoni Toker*,† †
Institute of Nanotechnology and Advanced Materials, Bar-Ilan University, Ramat-Gan 5290002, Israel Chemistry Department, Weizmann Institute of Science, Rehovot 978007, Israel § Spectrum Warfare Systems Department, NSWC Crane Division, Crane, Indiana 47522, United States ‡
ABSTRACT: Previous studies have shown that the gas-phase fragmentation of the retinal chromophore after S0−S1 photoexcitation results in a prominent fragment of mass 248 which cannot be explained by the cleavage of any single bond along the polyene chain. It was therefore theorized that the fragmentation mechanism involves a series of isomerizations and cyclization processes, and two mechanisms for these processes were suggested. Here we used isotope labeling MS− MS to provide conclusive support for the fragmentation mechanism suggested by Coughlan et al. (J. Phys. Chem. Lett. 2014, 5, 3195).
1. INTRODUCTION
2. RESULTS AND DISCUSSION
All known forms of vision rely on a single chromophorethe retinal protonated Schiff base (RPSB)as a photodetector.1,2 The RPSB has a tunable absorption, and acts as an optical switch: upon photon absorption, it undergoes a photoisomerization which initiates a series of transformations involving the chromophore and the surrounding protein. In the gas phase, once the molecule absorbs a photon or is collisionally heated, the internal energy cannot be transferred to the surroundings, and eventually causes the molecule to fragment, on very long time scales (ranging up to hundreds of microseconds).3 By measuring the photofragment yield as a function of the wavelength, one can thus measure the gas-phase absorption of the molecule. Such measurements have been instrumental in understanding the RPSB color-tuning mechanism.4 These measurements further indicated that the gasphase fragmentation mechanism of the RPSB involves a series of isomerizations and a cyclization process.4,5 This indirect evidence for gas-phase isomerizations of the chromophore has motivated direct measurements of gas-phase RPSB isomerizations, which were performed using ion mobility spectroscopy (IMS).6−9 Although the fragmentation process is not relevant for the function of the chromophore within the protein, it nonetheless poses an intriguing enigma: Why, of all the energetically available fragmentation channels, is this specific pathway chosen? Two mechanisms for the fragmentation process, illustrated in Figure 1, were suggested,5,6 and support for the second mechanism was given on the basis of a novel implementation of ion mobility spectroscopy. Here we verify these results using isotope labeling MS−MS.
The RPSB chromophore ion has a mass of 340. Toker et al.5 have been shown that upon high-energy collision-induced dissociation (CID) the chromophore has many fragmentation channels, many of which are simple to explain. For example the most prominent fragment corresponds to the loss of a methyl group (as peaks with masses 325 and 310) . However, photoninduced dissociation with a single visible photon, as well as lowenergy collision-induced dissociation, yields only one prominent charged fragment of mass 248. This fragment does not correspond to the cleavage of any single bond along the polyene chain, but does correspond to toluene emission from the central part of the molecule, accompanied by “tying” together both ends. Similar processes are known to occur in carotenoid fragmentation.10 Toker et al.5 (see Figure 1, path a) have suggested a mechanism by which this can happen, which involves a series of isomerizations and a Diels−Alder cyclization leading to a three-ring structure, followed by the emission of the section of the chromophore which lies between C9 and C13. Coughlan et al.6 have shown that the collisional crosssection of the RPSB photofragments corresponds to that of a βionone PSB (see Figure 1), and suggested a mechanism for its formation which involves the formation of an intermediate eight-membered ring and the emission of the section of the chromophore which lies between C10 and C15 (see Figure 1, path b). Here we test this hypothesis using isotope labeling and tandem mass spectrometry (MS−MS).
© XXXX American Chemical Society
Received: March 10, 2016 Revised: April 4, 2016
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DOI: 10.1021/acs.jpca.6b02525 J. Phys. Chem. A XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry A
Figure 1. RPSB fragmentation mechanism: (a) mechanism suggested by Toker et al.,5 (b) mechanism suggested by Coughlan et al.1
Figure 2. Fragment mass spectra following argon collision: (top) RPSB, (middle) derivative D1 labeled in positions 14 and 15, (bottom) derivative D2 labeled in positions 8 and 19.
The measurements were performed on a micromass Q-TOF spectrometer from Waters. We have measured the MS−MS of the regular RPSB (denoted here as R), as well as a derivative D1 in which the carbons at positions 14 and 15 were replaced by 13C, and a second derivative D2 which is labeled in positions 8 and 19. The retinals with specific 13C labels were synthesized by using previously described methods.11,12 We used CID with an argon buffer gas, and chose an intermediary collision energy of ∼50 eV, such that for R the mass 248 peak is dominant; however, other fragmentation pathways (which are dominant at higher collision energies) are visible. These include the emission of one, two, or three methyl groups leading to fragments of masses 325, 310, and 295, respectively, as well as fragments with masses below 200, which will be discussed below. The MS−MS results for the three samples are shown in Figure 2. The power of the isotope labeling method is that it allows us to identify which portion of the molecule is emitted from the chromophore, for each fragmentation pathway. For example, we see that the methyl loss peak (mass 325) for D2 is split into two peaks (masses 326 and 327), and from the relative heights
of the peaks, we conclude that in approximately one-third of the cases it is the methyl group at position C19 which is cleaved. If the peak at mass 310 for RPSB corresponds to two independent cleavages of a methyl group, we would expect that in approximately half of the cases one of the two lost methyls will be that in position C19. This is indeed the case, and we conclude that the fragments of masses 310 and 295 result from double and triple methyl losses rather than the loss of ethyl and propyl groups. The peak of mass 192 in R has shifted to mass 194 for sample D1 and to mass 193 for sample D2, indicating that this fragment corresponds to cleavage of the C8−C9 bond. Similarly, we can determine that the peak at mass 152 in R corresponds to cleavage of the C10−C11 bond, and the mass 126 peak corresponds to cleavage of the C12−C13 bond. In these three cases, the bond cleavage is accompanied by a hydrogen transfer. Additionally, we observe small peaks for R at masses 175 and 177which do not move in samples D1 and D2. These peaks do not correspond to any simple cleavage along the polyene chain, and must involve an intermediary cyclization stage. Nevertheless, we have not yet been able to determine the corresponding mechanism. These results point B
DOI: 10.1021/acs.jpca.6b02525 J. Phys. Chem. A XXXX, XXX, XXX−XXX
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The Journal of Physical Chemistry A
Dipolar Assisted Rotational Resonance NMR of Tryptophan and Tyrosine in Rhodopsin. J. Biomol. NMR 2004, 29, 11−20.
to an interesting interplay between fragmentation which follows cyclization (resulting in the main 248 peak, as well as in the smaller 177 peak) and between fragmentation mechanisms which do not involve cyclization (resulting in the peaks at masses 192, 152, and 126). The main finding of this work is that the main fragmentation peak has mass 248 for samples R and D1 and mass 250 for sample R. This proves that the fragment does correspond to the emission of the section of the RPSB between C10 and C15, in keeping with the mechanism suggested by Coughlan et al.,6 and demonstrating the power of the IMS technique for determining fragmentation mechanisms.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: 972-3-5317406. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was supported by GIF Research Grant No. I2370-303.7/2014 and by the Israel Science Foundation.
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REFERENCES
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DOI: 10.1021/acs.jpca.6b02525 J. Phys. Chem. A XXXX, XXX, XXX−XXX
