Investigation of Fragmentation of Tryptophan Nitrogen Radical Cation
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This work describes investigation of the fragmentation mechanism of tryptophan N-indolyl radical cation, H3N+-TrpN• (m/z 204) studied via DFT calculations and several gas-phase experimental techniques. The main fragment ion at m/z 131, shown to be a mixture of up to four isomers including 3-methylindole (3MI) π-radical cation, was found to undergo further loss of an H atom to yield one of the two isomeric m/z 130 ions. 3-Methylindole radical cation generated independently (via CID of [CuII(terpy)3MI]•2+) displayed gas-phase reactivity partially similar to that of the m/z 131 fragment, further confirming our proposed mechanism. CID of deuterated tryptophan N-indolyl radical cation (m/z 208) suggested that up to six H atoms are involved in the pathway to formation of the m/z 131 ion, consistent with hydrogen atom scrambling during CID of protonated Trp.
KeywordsTryptophan N-indole radical 3-Methylindole Quinolinium ion Collision-induced dissociation Ion-molecule reactions Density functional theory calculations
Radical ions of proteins, peptides, and amino acids have attracted considerable interest in the past decade [1, 2, 3, 4]. Understanding fragmentation mechanisms of peptide radical ions is crucial for interpreting experimental data in techniques like electron capture dissociation , electron transfer dissociation , and radical-directed dissociation . Among the various amino acid-based radicals, tryptophan is especially intriguing as it can form two types of biologically important radicals—the tryptophan π-radical cation (Trp•+) and the indolyl radical (TrpN•). Both types are believed to be involved in electron transfer processes  as well as in protein oxidation . Both types of Trp radicals were studied by tandem MS in peptide systems [10, 11, 12, 13].
When formed, this distonic ion formally has the spin localized in a sigma orbital on the N of the indole ring, but in reality it is delocalized over the π-system of the indole .
Chemicals and Reagents
All chemicals and reagents were used as received without any further purification. L-tryptophan, 3-methylindole, 2,2';6',2"-terpyridine (terpy), and tert-butyl nitrite, di-tert-butyl nitroxide were all purchased from Sigma-Aldrich (Milwaukee, WI, USA). Methanol and D2O were purchased from Fisher (Pittsburg, PA, USA). Water was purified (18 MΩ) in-house.
Mass spectrometry experiments were carried out at Northern Illinois University using a Bruker Esquire 3000 quadrupole ion trap mass spectrometer (Bruker Daltonics, Billerica, MA, USA) modified to conduct ion-molecule reactions as described previously [22, 23]. The N-nitrosylated tryptophan was generated by allowing a 1.5:1 mixture of tert-butylnitrite and a 1 mM solution of Trp (in 50/50 methanol/water with 1% acetic acid) to react for 10 min at room temperature. The reaction mixture was diluted a hundredfold using 50/50 methanol/water and introduced into the ESI source of the mass spectrometer at a flow rate of 4 μL min–1. The nebulizer gas, needle voltage, and temperature were adjusted to about 15 psi, 3.4 kV, and 200°C, respectively. Radical cations H3N+-TrpN• were produced by CID using collision energy sufficient to dissociate the majority of the precursor ions. The deuterated form of the radical was formed using the same procedure, except with D2O/CH3OD as the solvent.
For the production of 3-methylindole (3MI) π-radical cation, 200 μL of 1 mM 3MI stock solution (in 50/50 methanol/water) was mixed with 100 μL of 1:1 mixture of 1 mM CuSO4 and 2,2';6,2"-terpyridine stock solutions. The mixture was then diluted with 700 μL of methanol and immediately introduced into the ESI source of the mass spectrometer at a flow rate of 4 μL min–1. The nebulizer gas, needle voltage, and temperature were adjusted to 18 psi, 3.4 kV, and 200°C. The radical was obtained through the fragmentation of [CuII(terpy)3MI]•2+ ternary complex using collision-induced dissociation. The corresponding radical cations were then mass-selected with a window of 1 m/z for further analysis.
All calculations were carried out by the Gaussian09 package of programs . Geometry optimizations and harmonic vibrational frequency calculations were performed at B3LYP/6-311++G(d,p) level of theory [25, 26, 27, 28, 29, 30, 31]. In the case of open-shell systems, spin-unrestricted calculations (UB3LYP) were used. Intrinsic reaction coordinate (IRC) calculations  were employed on all the transition states, followed by geometry optimizations on the structures produced by the IRC calculations in order to ensure that the transition states were indeed connected to the appropriate reactant and product ions.
Results and Discussion
In our previous work, we calculated the barrier for the N-indolyl tryptophan radical cation (H3N+-TrpN•) rearranging into the lower energy π-radical cation to be 137 kJ mol–1 . Since the π-radical cation fragments under CID conditions to give the 3-methylindoleum ion at m/z 130 and there is very little m/z 130 present in the CID of H3N+-TrpN•, the fragmentation pathway must have a barrier that is lower than required for conversion into the π-radical. Earlier, we postulated that the base peak in the CID spectrum of H3N+-TrpN•, m/z 131, is the radical cation of 3-methylindole (3MI•+). In order to form this species, the initial ion at m/z 204 has to lose CO2 and NH = CH2 moieties. The process is initiated by a proton transfer from the NH3+ to C3 of the indole (Figure 1a). After a series of geometry changes through bond rotations (first about Cβ − Cγ, then Cα − Cβ and C − OH bonds, see Supplementary Figure S1 for details) the Cα − C bond cleaves followed by HAT from the C-terminus to the indole nitrogen atom resulting in the loss of CO2. This step of the mechanism is consistent with CO2 loss reported earlier by Knudsen and Julian  for C-terminal TrpN• in small peptides. After another hydrogen-atom transfer (HAT) from the N-terminus to Cα followed by Cα − Cβ bond dissociation, an isomer of the m/z 131 ion (1) is formed with an overall barrier of 129 kJ mol–1, slightly lower than that required for the formation of the π-radical cation by proton transfer (137 kJ mol–1). This higher energy isomer 1 of the m/z 131 ion can undergo a further 1,2-HAT to yield the lower energy structure, the 3MI•+ ion (1a). Both m/z 131 ions, 1 and 1a, can lose a hydrogen atom and form 3-methyleneindolium ion (m/z 130, 2a). Alternatively, ion 1 can undergo ring expansions leading to lower energy isomers with m/z 131 1b and 1c, either of which can lose a hydrogen atom and form quinolinium ion 2b at m/z 130. The overall barriers associated with forming 1a, 1b, and 1c from 1 are comparable suggesting that a mixture of isomers can be formed upon CID. The structures of the isomeric m/z 131 ions 1-1c displaying spin delocalization are shown in Figure 1b.
Collision-Induced Dissociation and Ion-Molecule Reactions
To evaluate individual steps of the theoretical mechanism experimentally, we examined the gas-phase reactivity of the ion m/z 131 formed by two ways. First, the m/z 131 ion was isolated in the trap after CID of H3N+-TrpN• (m/z 204). Independently, 3MI π-radical cation was formed via in-source dissociation of the ternary complex [CuII(terpy)(3MI)]•2+.
CID of m/z 131 ions independent of the way of their production resulted in a single product at m/z 130 (see Supplementary Figure S1) corresponding to the loss of one hydrogen atom. This is consistent with the proposed mechanism, but does not shed light on the structure of the m/z 131 ion(s) formed via CID of H3N+-TrpN•.
H/D Exchange Experiments
Thus, a total of six exchangeable hydrogen atoms (three from the N terminus, one from the C terminus, and two from positions C2 and C4 in the side chain) participate in the formation of the major fragment during CID of H3N+-TrpN•. A semiquantitative explanation of the observed experimental intensities of the ions in the m/z 131-134 cluster of Figure 3 is given in the Supplement (Supplementary Figure S4 and explanation below).
In conclusion, the fragmentation mechanism of tryptophan N-indole radical cation, H3N+-TrpN•, under low-energy CID was studied theoretically and experimentally. The main fragment ion, an m/z 131 ion, was shown to be a mixture of several isomers, the 3MI π-radical cation (1a) and the higher energy 3H,3–methyleneindolium ion (1) and the products of its ring expansion 1b and 1c; all of these isomeric ions undergo further loss of a hydrogen atom to yield either the 3-methyleneindolium (2a) or quinolinium (2b) ions at m/z 130. The various stages of the proposed mechanism were verified by CID, H/D exchange, and ion-molecule reactions. 3-Methylindole radical cation (1a) was also generated in an independent way and demonstrated gas-phase reactivity similar to a fraction of the m/z 131 ions from H3N+-TrpN•, thereby providing an extra support for our proposed mechanism.
The authors acknowledge support for this study by the Natural Sciences and Engineering Research Council (NSERC) of Canada and made possible by the facilities of the Shared Hierarchical Academic Research Computing Network (http://www.sharcnet.ca). Support from the Northern Illinois University is also gratefully acknowledged. The authors also thank Professor Frantisek Turecek (University of Washington) for useful suggestions on the fragmentation pathways.
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