Factors Affecting the Production of Aromatic Immonium Ions in MALDI 157 nm Photodissociation Studies
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Immonium ions are commonly observed in the high energy fragmentation of peptide ions. In a MALDI-TOF/TOF mass spectrometer, singly charged peptides photofragmented with 157 nm VUV light yield a copious abundance of immonium ions, especially those from aromatic residues. However, their intensities may vary from one peptide to another. In this work, the effect of varying amino acid position, peptide length, and peptide composition on immonium ion yield is investigated. Internal immonium ions are found to have the strongest intensity, whereas immonium ions arising from C-terminal residues are the weakest. Peptide length and competition among residues also strongly influence the immonium ion production. Quantum calculations provide insights about immonium ion structures and the fragment ion conformations that promote or inhibit immonium ion formation.
Key wordsImmonium ions Photodissociation Peptide ion fragmentation Aromatic residues
Immonium ions are small, single amino acid structures that result from peptide fragmentation in tandem mass spectrometry. The form of these ions is [NH2=CHR]+, where R is an amino acid side chain. Immonium ions associated with aromatic residues are particularly abundant . High energy collision-induced dissociation (CID) was originally used to study these ions [1, 2], and various mechanisms of formation have been proposed [3, 4, 5, 6]. For example, an N-terminal immonium ion can form from a rearrangement in the oxazolone ring of a b-type ion. An internal immonium ion can be created by first forming an a-type ion and subsequently releasing the N-terminal side of the ion . Baldwin et al. found that factors such as location within the peptide influence the intensity of the immonium ion signal in mass spectra. They reported that the ion signals are strongest when the corresponding amino acid is located at the N-terminus and lowest at the C-terminus of peptides without an arginine or lysine . Additionally, a competition between other residues within the peptide was discerned.
Since their observation can validate residues within a peptide, immonium ions are also relevant to peptide identification programs [7, 8]. For example, this might contribute to the analysis of antibodies. The complementarity determining regions (CDRs) of antibodies often contain hydrophobic residues, particularly those with aromatic side chains, which are involved in antigen binding . Since aromatic residues produce abundant immonium ions, the observation of these small fragment ions can facilitate the recognition of CDR peptides.
In the present research, a matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometer generates predominately singly charged ions. This instrument has been modified to achieve high energy fragmentation with a 157 nm vacuum ultraviolet (VUV) photodissociation laser . Photodissociation provides an excellent opportunity to investigate trends associated with immonium ion formation because these ions are formed in abundance . Ions associated with the aromatic residues histidine, tryptophan, tyrosine, and phenylalanine are the focus of this work. Variations in immonium ion yield due to position within a peptide, peptide length, and compositional effects are all examined. Additionally, theoretical calculations offer insights about the stability of various fragment structures and likely formation mechanisms.
In addition to those directly synthesized, some peptides were obtained by digesting proteins with trypsin. Rituximab, a monoclonal antibody, was first reduced with 0.1 M dithiothreitol for 1 h at 56 °C. The reduced cysteines were alkylated with 0.1 M iodoacetamide in the dark for 30 min. The reaction was quenched with the addition of more dithiothreitol and spin filtered with ammonium bicarbonate solution to remove reagents. The antibody was then digested with trypsin (80% ammonium bicarbonate and 20% acetonitrile solution) in a Pressure BioSciences Inc. (South Easton, MA, USA) barocycler for 99 cycles at 37 °C in order to accelerate the digestion process. Each cycle was 50 s at 20 kpsi and 10 s at atmosphere. Guanidination labeling of lysines with S-methylisothiourea was performed to improve MALDI ionization . A peptide library used in previous research contained peptides derived from tryptic digestion of ribosomal proteins [13, 14].
157 nm Photodissociation
For each synthesized peptide, 1 μL of approximately 500 μM peptide solution was deposited with 1 μL of 10 mg/mL of α-cyano-4-hydroxycinnamic acid in 50:50 water:acetonitrile with 0.1% trifluoroacetic acid. All antibody protein digests underwent reversed-phase liquid chromatography with an Eksigent Nano 2D LC coupled to an Eksigent Ekspot MALDI spotter (Framingham, MA, USA). This spotting procedure served to reduce the number of peptides per spot, minimizing ionization competition among peptides. A self-packed C18 capillary column was used to separate the mixture. The eluent was then mixed with matrix and spotted onto a MALDI plate.
As previously described, an ABI 4700 MALDI TOF-TOF mass spectrometer (Framingham, MA, USA) was modified to pass 157 nm VUV light from a Lambda Physik CompexPro laser (Santa Clara, CA, USA) through its collision cell . The laser requires 5% fluorine in helium to produce the 157 nm light. As ions pass through the collision cell, they are irradiated with one light pulse. Resulting spectra include both PSD and photodissociation fragments. In Table 1, peptides 9–40 were analyzed on an ABI 4800 MALDI TOF-TOF mass spectrometer that was modified in a similar manner. Data from 1000 laser shots were averaged to create each spectrum.
Data Explorer 4.6 software is provided with the ABI 4700 MALDI-TOF/TOF instrument. This software can calculate the percent abundance of each fragment relative to the height of the base peak. For the synthesized peptides, the relative percent abundance was averaged over two spectra, which improved data reproducibility. This analysis was also performed by calculating peak heights as a percentage of the total fragment ion intensity. Samples from the peptide library with low S/N were excluded from the data set.
Where ∆G(gas) = change in gas phase free energy; ∆H(gas) = change in gas phase enthalpy; T = temperature (298.15 K); ∆S(gas) = change in gas phase entropy; ∆E(SCF) = self-consistent field energy, i.e., “raw” electronic energy as calculated at the triple-ζ level; ∆ZPE = change in vibrational zero point energy.
Here E(gas) is the gas-phase electronic energy, which is zero for a proton but non-zero for the hydrogen atom. The 5/2(RT) term is also included in estimations of reaction enthalpies. The translational entropy (S) for the hydrogen atom and proton was calculated as 26.04 eu using the Sackur-Tetrode equation.
To properly evaluate the energy of different structures, we attempted to locate the lowest energy conformer for each species, or at least one of the lowest energy conformers. The structures determined through our geometry optimizations only confirmed that we have obtained local minima, which will not necessarily be the global minima. We partially addressed this by using the Global-MMX program (GMMX) as employed by PCModel . This conformational analysis employed the MMX force field  and generated a large number of reasonably low energy conformers. Although this analysis is useful for generating a small subset of reasonable structures, these structures and energies were not accurate enough for our analysis. These structures were therefore re-optimized with DFT. The gas phase free energy was calculated for a small set of the lowest energy DFT optimized structures (based on electronic energy at the double-ζ level), and the structure with the lowest gas phase free energy was then taken to be the global minimum. This procedure was employed for all structures except in instances where the number of structures to examine was particularly large. Conformational searches for open-shell species were performed on similar closed-shell species, and these cases are discussed in more detail in the Electronic Supplementary Material.
Results and Discussion
A complication associated with the experiments just described is that the yield of immonium ions from a particular type of residue depends not only on the position of that residue in the peptide but also on what other residues are present in the sequence. As will be discussed in detail below, other aromatic residues within a peptide can compete for the available proton, thereby influencing the observed immonium ion intensities. In order to reduce this effect, peptides 9–40 were synthesized with only a single aromatic residue in various positions. The trends displayed in Figure 3c and d are similar to those in Figure 3a and b. The position of an aromatic residue strongly affects its immonium ion intensity.
Calculated proton affinities (PA) and gas-phase basicities (GB) of relevant immonium ions. Not all of these immonium ions were detected experimentally, but were calculated to provide a point of reference. Unless indicated otherwise, the protonation site is the imine. All values reported in kcal/mol
Peptides with an N-Terminal Arginine
Immonium and a2 Ions
a2 Ions behave somewhat differently from a4 and a6 ions. As seen in Supplemental Figures S4 and S5, the a2 ion pathway is more favorable than any other a-type ion pathway and with nearly all aromatic residues, the a2 ion is more intense than the corresponding immonium ion. This increased a2 ion intensity suggests that its stability is enhanced, possibly due to a five-membered cyclic structure [34, 35, 36]. A strong a2-NH3 ion may result from an increased stability of the arginine side chain forming a seven-membered ring with the N-terminus .
Calculated Thermodynamics (ΔG) for Various Decomposition Reactions of a2 +1 ions. All values reported in kcal/mol
a2 + 1 ion
A second potential reason why a2 + 1 ions may decay to a2 ions rather than immonium ions is that during 157 nm photodissociation, a peptide absorbs a 7.9 eV photon and fragments. If an a2 + 1 ion is formed, this ion could contain less internal excitation energy than a larger ion, preventing the immonium ion pathway. Formation of an a-type ion requires breaking a C–H or N–H bond , whereas formation of the immonium ion requires breaking the conjugated amide bond. Another possibility is that there is competition with the arginine at the N-terminus. A pathway described previously to an a1 ion indicates that proton affinity plays a critical role in determining what ion will be formed . Since arginine has a higher proton affinity than the aromatic residues, an arginine a1 ion would be more favorable than the aromatic immonium ion. When aromatic residues are at other positions in these peptides, less intense a-type ions are observed, consistent with the more facile production of the immonium ion.
Peptides with a C-Terminal Arginine
Aromatic Residues at N- or C-Terminus
For peptides with a C-terminal arginine, immonium ions formed from aromatic residues at the N-terminus exhibit somewhat diminished intensities compared with positions 3 and 5. These a1 ions are not formed if the proton is sequestered on the C-terminal arginine. For histidine, this is less of a problem since the imidazole ring contains a basic protonation site that leads to the formation of strong a2 and b2 ions. As in the earlier discussion of aromatic residues next to N-terminal arginines, formation of these two ions potentially competes with formation of histidine immonium ions. For tryptophan, tyrosine, and phenylalanine, these residues lack a basic protonation site in their side chains, so the N-terminal amine will be the preferential protonation site . If an a + 1 ion is the initial step to form this immonium ion, this fragment must contain the proton. Alternatively, immonium ions from internal residues initially form a + 1 or x + 1 radical ions. These radical ions are probably vibrationally excited with a mobilized proton that can form immonium ions more readily.
The reduced intensity for an immonium ion derived from an aromatic residue at the C-terminus likely involves several factors. Eight-residue peptides having N-terminal arginine do not form a8 ions. Consequently, C-terminal immonium ions would not form from a-type ion precursors. A mechanism for forming a phenylalanine immonium ion from a y1 ion has been proposed . However, if arginine is at the N-terminus, relatively few y-type ions will be formed and particularly few y1 ions. Again, the only exception would be histidine, which is basic enough to form an intense y1 ion. DFT calculations on the lowest energy y1 ions show that histidine can be stabilized by an intramolecular H-bond between the imidazole and the ammonium ion; however, there are no other significant structural features in the other y1 ions. Because the protonation site of histidine is in its side chain imidazole, the interaction proposed by Harrison and coworkers to facilitate immonium ion formation will be unlikely, consistent with the weak immonium ion even for this residue . Similarly, an x1 + 1 ion would likely be unable to form an immonium ion. This ion would have the same hydrogen bonds as the y1 ion that inhibits loss of the carboxylic acid to form the immonium ion.
To further support the observation of low intensity immonium ions from an amino acid at the C-terminus of a peptide, the arginine immonium ion at 129 Da was also investigated for peptides 1–8. However, this ion is typically weak with an average abundance of 5% relative to the base peak when arginine is at the N-terminus or less than 2% when at the C-terminus. Another arginine associated ion is at 112 Da, which is formed by the loss of ammonia from the immonium ion. The 112 Da ion is typically stronger than the immonium ion at 129 Da because a stable seven-membered ring is formed by losing ammonia . When arginine is at the N-terminus, the average relative abundance of the 112 Da peak is 41%, and at the C-terminus, the abundance is 13%. With arginine at the C-terminus, y1 ions with considerable intensity are seen in Supplemental Figure S2. However, the loss of COOH needed to proceed to the immonium ion is likely inhibited by hydrogen bonds from the backbone carboxylic acid to the side chain guanidine group . Additionally, this pathway is unlikely, which is consistent with histidine as noted above because both residues contain a side chain protonation site.
Peptide Composition Dependence
As mentioned above, the presence of multiple aromatic residues in a single peptide can affect the immonium ion production. Figure 3a and b display data from peptides that each contained four aromatic residues, whereas Figure 3c and d show results for peptides with only a single aromatic residue. The latter two figures display a factor of two increase in the percent total fragment ion intensity for all immonium ions, as a result of reducing the competition with other residues. For example, the histidine immonium ion intensity from peptides with arginine at the C-terminus ranges from 4.7% to 6.7% in Figure 3a, but increases to 12.7%–18.2% in Figure 3c. In peptides with arginine at the N-terminus, the values range from 3.1%–6.3% in Figure 3b and increase to 6.1%–10.0% in Figure 3d. Similar results are obtained for the other aromatic residues. A second example of competition among residues for formation of immonium ions can be seen by comparing Figure 3c and d. The relative intensities of histidine, tryptophan, tyrosine, and phenylalanine immonium ions tend to be higher when arginine is at the C-terminus. As discussed in previous sections, when arginine is at the N-terminus, formation of a strong 112 Da ion competes with formation of aromatic immonium ions.
In order to further corroborate competition among residues, we investigated whether the peptides that produce the largest immonium ion signals are those with a single aromatic residue. In our 238 peptide library, 110 peptides contained tyrosine and 57 (51.8%) of these contained another aromatic residue. However, only three of the 21 peptides (14.3%) that produced an intense tyrosine immonium ion (above 50% of the base peak) contained another aromatic residue. Likewise, of the 121 peptides that contained phenylalanine, 60 (49.6%) contained another aromatic residue. However, of the 14 peptides that produced an intense phenylalanine immonium ion, only 1 (7.1%) contained another aromatic residue. This means that an intense tyrosine or phenylalanine immonium ion is less likely if another aromatic residue is in the sequence. The peptides in which tyrosine is present with another aromatic residue and still produces above 50% relative abundance are EFQTYFR, YAAHDENNEYK, and MESSAGTGFYYTTTK. For phenylalanine, the peptide is EFQTYFR. Although all four of these peptides contain another aromatic residue, they either contain two phenylalanine or tyrosine residues, or they have limited competition from the other residues within the sequence. Histidine and tryptophan were not included in this analysis because histidine typically has the most intense immonium ion regardless of peptide composition and there were not enough tryptophan containing peptides.
Having systematically investigated the factors that influence immonium ion intensity, we now reconsider data presented above for angiotensin-I and bradykinin 2-9. Based on their two sequences, DRVYIHPFHL and PPGFSPFR, the biggest contribution to the difference in phenylalanine immonium ion intensity is the sequence composition. Since the phenylalanines are located in the middle of the sequence, they do not suffer from the effects of being located at either termini or C-terminal to an arginine. The lengths of these peptides only differ by two residues, so no major contribution is expected from a length effect. The major difference is the sequence composition because angiotensin-I contains one phenylalanine, two histidines, and one tyrosine. With the presence of these other aromatic residues, the competition for producing a phenylalanine immonium ion is strongly impeded. On the other hand, bradykinin 2-9 contains only two phenylalanines and no other aromatic residues, so there is little competition with other amino acids.
A number of factors affect immonium ion intensity in 157 nm photodissociation experiments. Residue position, peptide length, and peptide composition were studied by synthesizing a variety of peptides and collecting peptides from tryptic digests of various proteins. It was found that residue position highly affects the immonium ion intensity. The positions of aromatic residues relative to other amino acids, such as arginine, are also an important factor that affects immonium ion intensity. Smaller peptides form more intense immonium ions than larger peptides. Finally, other residues within the peptide can reduce the intensity of an immonium ion because of competition for ionization. The results presented here will assist in identifying peptides that contain aromatic residues. An example would be peptides that contain the CDRs of antibodies.
Although early high-energy CID reports described abundant production of immonium ions [1, 2, 3, 4, 5, 6], current proteomics experiments utilize low-energy CID, in which these ions are rarely observed both because they are not produced in abundance and because most experiments are performed with ion traps that have a low mass cut-off. In contrast, photodissociation in a MALDI-TOF/TOF mass spectrometer produces a plethora of detectable immonium ions. A future experiment that should be performed to further investigate these factors is to vary the intensity of the 157 nm laser. The abundance of each of the aromatic immonium ions might change as a result of increasing or decreasing the laser energy. Absorption of multiple photons can occur at higher laser energies that will increase the number of fragmentation events and complicate the trends observed in these experiments.
The authors thank NIH and NSF for financial support under grants R01 GM103725 and CHE-1012855. M.H.B. thanks the Institute for Basic Science in Korea for support of this work (IBS-R10-D1).
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