Leveraging Electron Transfer Dissociation for Site Selective Radical Generation: Applications for Peptide Epimer Analysis
Traditional electron-transfer dissociation (ETD) experiments operate through a complex combination of hydrogen abundant and hydrogen deficient fragmentation pathways, yielding c and z ions, side-chain losses, and disulfide bond scission. Herein, a novel dissociation pathway is reported, yielding homolytic cleavage of carbon–iodine bonds via electronic excitation. This observation is very similar to photodissociation experiments where homolytic cleavage of carbon–iodine bonds has been utilized previously, but ETD activation can be performed without addition of a laser to the mass spectrometer. Both loss of iodine and loss of hydrogen iodide are observed, with the abundance of the latter product being greatly enhanced for some peptides after additional collisional activation. These observations suggest a novel ETD fragmentation pathway involving temporary storage of the electron in a charge-reduced arginine side chain. Subsequent collisional activation of the peptide radical produced by loss of HI yields spectra dominated by radical-directed dissociation, which can be usefully employed for identification of peptide isomers, including epimers.
KeywordsPhotodissociation RDD ECD Isoaspartic acid
Radical chemistry has played an important role in the development of mass spectrometry over the years, and the combination continues to evolve and produce useful applications. Unfortunately, many modern ionization sources are geared towards gentle transfer of large biomolecular ions into the gas phase, and do not typically generate odd-electron ions in significant abundance [1, 2]. Methods for introducing radicals post-ionization are therefore required. Several options are available, including photodissociation  and collisional activation-based approaches [4, 5, 6]. Radical chemistry can also be combined with ion–ion reactions utilizing either activation method . Once created, odd-electron chemistry has many uses in the gas phase, including cross-linking , examining three-dimensional structure , accessing novel dissociation pathways [10, 11], and isomer identification . Radical-directed dissociation (RDD) is often sensitive to fine structural details because dissociation is preceded by radical migration, which is guided by the relative orientation of hydrogen atom(s) that must be abstracted to allow relocation of the nascent radical to the ultimate site of fragmentation .
A key for many of these applications is site-specific generation of a radical via homolytic photodissociation of labile bonds. Dissociation occurs due to electronic excitation to dissociative excited states, leading to fragmentation on the femtosecond timeframe . Many bonds have been demonstrated to be suitable, including carbon–iodine, carbon–sulfur, and sulfur–sulfur . In optimal cases, radical yields can be quantitative with a single laser pulse , but implementation requires coupling an ultraviolet laser to a mass spectrometer—an experimental configuration that is not currently available commercially. The need for customized instrumentation has likely limited accessibility to the technique.
In contrast, electron-transfer dissociation (ETD) is another radical-based fragmentation method that is widely available on many commercial platforms. ETD was developed following discovery of electron capture dissociation (ECD) , and both facilitate fragmentation by delivering an electron to a multiply charged cation, often yielding very similar results. In ETD, the electron originates from a molecular anion and transfer results in charge reduction, energy increase, and dissociation. For peptides, fragmentation into c/z ions typically dominates, with the side benefit that post-translational modifications (PTMs) are often preserved . ETD can also be used to identify isomers, such as iso-aspartic acid, for which a unique fragment ion is generated . ECD has been used to interrogate three-dimensional protein structure by mapping out the relative strength of noncovalent interactions between sequence remote regions . Thus, there are many similarities between applications of RDD and ETD/ECD. Interestingly, electron transfer also converts an ion into a hydrogen abundant radical, which can spontaneously convert into a hydrogen deficient radical, the same type of radical operative in RDD. Indeed, some of the fragmentation observed in ETD/ECD is attributable to RDD , but precise mechanisms accounting for all observed fragmentation in ETD/ECD are not universally agreed upon and have been the source of significant study and controversy .
In this work, we present a method for generating site-specific cleavage of carbon–iodine bonds via ETD rather than photodissociation. Both homolytic cleavage due to excitation of dissociative electronic states yielding loss of iodine radical (–I•), and loss of iodine accompanied by hydrogen (–HI) are observed. Interestingly, the ratio of these two losses varies as a function of additional collisional activation, with the amount of –HI loss increasing dramatically with greater activation for some peptides. A novel electron storage mechanism involving the side chain of arginine, which allows decoupling in time of electron transfer and dissociation, is proposed to account for these observations. Density-functional calculations support the proposed mechanism. Finally, the utility of ETD-generated radicals for isomer identification is illustrated by examination of several peptide isomers, dissimilar in structure due to isomerization or epimerization at a single residue. For these applications, the ability to simultaneously generate both even- and odd-electron species is shown to be beneficial in some cases.
Organic solvents were purchased from Sigma Aldrich (St. Louis, MO, USA) or Fisher Scientific and used without further purification. Water was purified to 18.2 MΩ by a Millipore 147 (Billerica, MA, USA) Direct Q system. Amino acids and resins were purchased from Anaspec (Fremont, CA, USA). Trypsin and urea were purchased from Sigma Aldrich (St. Louis, MO, USA).
Peptide and Radical Precursor Synthesis
All synthetic peptides were synthesized manually using standard FMOC procedures with Wang, and Rink-Amide resins as the solid support . N-hydroxysuccinimide (NHS) activated iodobenzoyl esters were synthesized by following a previous procedure . Chloramine-T, sodium metabisulfite, and sodium iodide were purchased from Fisher Chemical (Fairlawn, NJ, USA). Peptides were iodinated by modification of a previously published procedure .
Mass spectrometric analysis was performed using an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Solutions containing ∼ 1 μM of protein in 49.5:49.5:1 MeCN/H2O/AcOH were directly infused using the Nanospray Flex Ion source (Thermo Scientific) at 2 uL per min through a 500 uL syringe. Ten μm i.d. emitters were pulled using a laser-based micropipette puller (Sutter Instrument, Novato, CA, USA). Higher energy collision induced dissociation (HCD) was used for the supplemental activation (from 0 to 35 normalized collisional activation) in ETD, which is termed EThcD. For ETD and EThcD experiments, 100 ms was used for fluoranthene reagent and peptide ion reaction time. The isolation window used for each step was 2–3 m/z to maximize ion count. For ETD-CID experiments, ETD or EThcD was first performed to generate the –I/–HI losses; then these peaks were isolated and subjected to collision induced dissociation (CID) of appropriate energy.
To quantify isomer identification, an Rchiral value approach, originally reported by Tao et al., was utilized . In this paper, Risomer represents the ratios of the relative intensities of a pair of fragments that varies the most between two isomers (RA/RB). If Risomer = 1, then the two mass spectra are the same and the species are not distinguishable. If Risomer >1, a larger number indicates a better degree of recognition. CID and RDD values were calculated using the +1 precursor charge state whereas ETD-CID was performed on the +2, as charge reduction to the +1 occurs before dissociation.
Theoretical calculations were carried out using density functional theory as implemented in Gaussian09 . The B3PW91 functional was used with the following basis sets: LANL2DZ  for iodine loss, 6-31G+(d) for disulfide cleavage, 6-31G(d) for c/z dissociation. Transition states were found using the QST3 approach and verified by calculating vibrational frequencies, for which a single imaginary value was obtained. The transition state for electron transfer to the disulfide functionality was not well behaved, and was therefore approximated via a step-wise scan of S–S bond length with all other coordinates relaxed. Time-dependent density functional theory at the B3LYP/LANL2DZ level was used to optimize the lowest-energy excited state of iodobenzene.
Results and Discussion
The loss of neutral iodine is also observed in Figure 1a and is more difficult to explain because, as mentioned above, direct interaction of the electron with the iodobenzyl moiety should lead to dissociation of negatively charged iodide, not neutral iodine. Furthermore, iodide anion would not be expected to relinquish an electron back to the peptide. TDDFT calculations on iodobenzene reveal the first excited electronic state has HOMO→LUMO transition character, see Figure 1c. Relaxation on this excited state via geometry optimization yields dissociation along the C–I bond and prompt loss of neutral iodine. This same pathway is frequently accessed in photodissociation experiments, where the dissociative state is populated via absorption of a UV photon . In order to account for the neutral iodine loss in ETD, we propose that electron transfer leads to temporary occupation of an excited state by the excess electron, which subsequently decays to a lower energy state while simultaneously exciting an electron in the iodobenzyl group, prompting homolytic cleavage of the C–I bond. This type of energy coupling between separate excited state electrons has been observed previously in the context of energy transfer .
Intrigued by the idea that radical arginine side chain could initiate delayed HI loss in ETD/ECD, we examined other well-known dissociation pathways. Calculations reveal that radical arginine side chain can also initiate c/z backbone fragmentation and disulfide bond cleavage via pathways analogous to the one shown in Scheme 2. Complete details, including energetics and structures, are shown in SI Figures S3 and S4. Backbone cleavage to yield c/z ions is slightly endothermic, though still possible with supplemental collisional activation. Disulfide bond fragmentation is exothermic and exposed by a minimal ~30 kJ/mol transition state. This pathway may contribute to the favored cleavage of disulfide bonds observed previously in ETD/ECD . These reaction pathways may yield some products observed following supplemental activation of ETnoD products.
The activation of carbon–iodine bonds by ETD as described above can be used to generate radicals site-specifically, enabling structurally sensitive RDD experiments that have advantages for isomer identification as described in the Introduction. In the past, these experiments were only possible by photoactivation of C–I bonds, but now it is clear that ETD may be used as well. Although in photodissociation-based experiments the fragment of interest is the loss of iodine, the equivalent radical generated by ETD is obtained by loss of HI. The difference occurs because electron transfer to a multiply protonated peptide yields a hydrogen abundant, odd-electron species. Loss of radical iodine from this odd electron ion would, therefore, yield an even-electron product. However, loss of HI, which is even-electron, maintains the radical on the peptide. To test the structural sensitivity of fragmentation initiated by ETD-generated radicals, we conducted a series of experiments on peptide isomers, including epimers.
Resulting Risomer Values from the Comparison of Different Peptide Epimers/Isomers
The Risomer values obtained from a variety of different activation methods for several peptides are listed in Table 1. The ETD-CID method is not only competitive, but actually yields the highest value for 2/4 of the examined peptides. The high structural sensitivity derives from the ability of ETD-CID to leverage structural discrimination from both even and odd electron processes simultaneously.
ETD can be used to activate carbon–iodine bonds in a laser-free experiment, enabling access to RDD-like spectra. Observation of neutral iodine loss strongly indicates excited electronic states not populated directly by the transferred electron can be generated in ETD. Delayed loss of HI suggests that arginine is capable of “storing” the electron temporarily, allowing a variety of ETD-like fragmentation pathways to be accessed following subsequent additional collisional activation. ETD-generated radicals are capable of identifying peptide epimers, at times with greater sensitivity than other methods. The generation of both even- and odd-electron products, which can be examined simultaneously, is a unique advantage of the ETD-activation method. It is clear that ion–ion reactions and radicals have many yet undiscovered uses that will continue to augment the capabilities of mass spectrometry in years to come.
The authors thank the NIH for financial support (NIGMS grant R01GM107099).
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