Covalent and Non-covalent Binding in the Ion/Ion Charge Inversion of Peptide Cations with Benzene-disulfonic Acid Anions

Abstract

Protonated angiotensin II and protonated leucine enkephalin-based peptides, which included YGGFL, YGGFLF, YGGFLH, YGGFLK, and YGGFLR, were subjected to ion/ion reactions with the doubly deprotonated reagents 4-formyl-1,3-benzenedisulfonic acid (FBDSA) and 1,3-benzenedisulfonic acid (BDSA). The major product of the ion/ion reaction is a negatively charged complex of the peptide and reagent. Following dehydration of [M+FBDSA-H]⁻ via collisional induced dissociation (CID), angiotensin II (DRVYIHPF) showed evidence for two product populations, one in which a covalent modification has taken place and one in which an electrostatic modification has occurred (i.e., no covalent bond formation). A series of studies with model systems confirmed that strong non-covalent binding of the FBDSA reagent can occur with subsequent ion trap CID resulting in dehydration unrelated to the adduct. Ion trap CID of the dehydration product can result in cleavage of amide bonds in competition with loss of the FBDSA adduct. This scenario is most likely for electrostatically-bound complexes in which the peptide contains both an arginine residue and one or more carboxyl groups. Otherwise, loss of the reagent species from the complex, either as an anion or as a neutral species, is the dominant process for electrostatically-bound complexes. The results reported here shed new light on the nature of non-covalent interactions in gas-phase complexes of peptide ions that can be used in the rationale design of reagent ions for specific ion/ion reaction applications.

INTRODUCTION

Tandem mass spectrometry has become the method of choice in generating sequence information from peptides and proteins.^{[1, 2, 3]} The major considerations for structural characterization via tandem mass spectrometry are ion-type and dissociation method.^[4] Typically, the ion-type is determined by the ionization method;^[5] however, manipulation of the ion-type in the gas-phase can be achieved via ion/electron,^[6] ion/molecule,^{[7, 8]} and ion/ion reactions.^{[9, 10]} Gas-phase ion/ion reactions allow for the decoupling of the ion-type from the ionization method by enabling the facile transformation of one ion-type into another within the mass spectrometer. One class of ion/ion reactions is charge inversion, where the ion of interest is produced in one polarity and then transformed to the opposite polarity.^{[11, 12, 13]} There have been several approaches to charge inversion, which include multiple ion transfer,^{[14, 15]} electrostatic adduct formation,^{[13, 16]} and covalent adduct formation.^{[17, 18]} Charge inversion of a peptide cation by selective modification of a primary amine has been demonstrated via Schiff base formation using an aldehyde containing reagent.^{[17, 18]} Charge inversion via electrostatic adduct formation has also been demonstrated on peptides using sulfonate-containing dianions, which display a propensity to form relatively stable long-lived complexes.^[13]

The nature of the analyte ion transformation that takes place in an ion/ion reaction is highly dependent upon the chemical characteristics of both the analyte and reagent ions.^[5] It has recently been demonstrated that selective covalent bond formation can take place in a gas-phase ion/ion reaction via Schiff base formation^[19] and via amide bond formation using N-hydroxysuccinimide ester-based reagents.^[20] Thus far, all ion/ion reactions that have resulted in covalent bond formation have involved multi-functional reagents. That is, the reagents have one or more groups that give rise to a strong electrostatic interaction to facilitate the formation of a relatively long-lived complex as well as one or more reactive groups. For example, Schiff base formation involving primary amines in peptides has been demonstrated using anions derived from 4-formyl-1,3-benzenedisulfonic acid (FBDSA) as reagents. A charged sulfonate group in the reagent anion strongly interacts with a protonated site on the peptide, thereby facilitating the formation of a long-lived complex. Schiff base formation within the complex involves the reaction of an uncharged primary amine (e.g., the N-terminus or ϵ-NH₂ side chain of lysine) with the aldehyde functionality of singly or doubly-deprotonated FBDSA. Upon collisional activation, nucleophilic attack on the carbonyl carbon of the aldehyde by an unprotonated primary amine leads to imine formation and loss of water. In the case of a singly protonated peptide in reaction with doubly deprotonated FBDSA, the process results in charge inversion of the peptide cation via covalent attachment (i.e., Schiff base formation), as indicated in process (1):

$${[\mathrm{M}+\mathrm{H}]}^{+}+{[\text{FBDSA}-2\mathrm{H}]}^{2-}\to {[\mathrm{M}+\text{FBDSA}-\mathrm{H}]}^{-}\to {[\mathrm{M}+♦]}^{-}+{\mathrm{H}}_2\mathrm{O}$$ (1)

The diamond symbol (♦) represents the addition of FBDSA to the peptide with a loss of a water molecule. When the diamond symbol is present with fragment ions, this represents the mass shift associated with a covalent modification.

The original paper describing Schiff base formation upon charge inversion involving a protonated peptide and doubly-deprotonated FBDSA showed fragmentation of the [M+♦]⁻ ion that gave a preponderance of evidence for Schiff base formation, with one exception. A y₇♦-ion, along with many abundant b♦-ions, was noted for the [M+♦]⁻ species generated by charge inversion of protonated angiotensin II (DRVYIHPF).^[17] The y₇-ion is, by far, the dominant product ion in the ion trap CID of the [M-H]⁻ ion, while no b-type ions are observed, but this fragment contains no primary amine groups that might be expected to undergo covalent modification. This exceptional case was speculated to result from a sub-population of ions that undergo strong electrostatic binding with the reagent without undergoing covalent reaction and that the loss of water for these ions is unrelated to Schiff base formation. Subsequent work with tryptic peptides also showed formation of modified ions with no obvious sites for Schiff base formation in some, but not all, cases.^[18] Furthermore, the observation of y_n+FBDSA-ions in those peptides that showed unexpected y♦-ions gave additional evidence for strong electrostatic interactions that survive collisional activation, even when amide bonds are cleaved.

In this work, we examine the origins of the product ions generated from singly protonated peptides that undergo charge inversion with doubly deprotonated FBDSA to shed light on the origins of those products that are inconsistent with Schiff base formation. For example, we clarify the nature of the electrostatic interaction involved in the negatively charged charge inversion product (i.e., interaction between sulfonate and protonated basic sites, such as arginine, ^{[21, 22, 23]} and/or negatively charged proton-bound dimer between the sulfonate and the C-terminus or side-chains of Asp or Glu),^{[13, 16]} as well as the nature of amino acid side chains in determining the likelihood for the observation of non-Schiff base dissociation products. To examine the behavior of species with strong electrostatic binding without the possibility for covalent reactions, we compare data collected for FBDSA reagent dianions with data collected using doubly deprotonated benzene disulfonic acid (BDSA). The latter species have been demonstrated to charge invert peptides and proteins via adduct formation (see reaction (2)).^[13]

$${[\mathrm{M}+\mathrm{H}]}^{+}+{[\text{BDSA}-2\mathrm{H}]}^{2-}\to {[\mathrm{M}+\text{BDSA}-\mathrm{H}]}^{-}$$ (2)

These reagent ions have been studied in reactions with model peptides angiotensin II and YGGFLX, where X is F, K, R, or H, and variations thereof. These studies have provided a deeper understanding of the nature of the non-covalent reactions that contribute to stabilizing ion/ion reaction complexes.

EXPERIMENTAL SECTION

Materials

Methanol, glacial acetic acid, acetic anhydride, and ammonium hydroxide were purchased from Mallinckrodt (Phillipsburg, NJ). Ammonium bicarbonate, acetyl chloride, 1,3- benzenedisulfonic acid, 4-formyl-1,3-benzenedisulfonic acid, angiotensin II, and YGGFL were purchased from Sigma-Aldrich (St. Louis, MO). The peptides YGGFLK, YGGFLF, and YGGFLH were purchased from CPC Scientific (San Jose, CA). The peptide YGGFLR was purchased from Pepnome Ltd. (Zhuhai City, China). All peptides were used without further purification. The procedures for N-terminal acetylation and C-terminal methyl esterification have been previously described.^[24] Peptide analytes were prepared at a concentration of ~150 µM in a solution of 50/50 (v/v) water/methanol prior to positive nanoelectrospray ionization (nESI). The anion reagent, FBDSA or BDSA, was prepared at a concentration of ~3.5 mM in a solution of 49.5/49.5/1 (v/v/v) water/methanol/ammonium hydroxide for negative nESI.

Mass Spectrometry

All experiments were performed on a 4000 QTRAP QqQ mass spectrometer (AB Sciex, Concord, ON, Canada), which has been modified for ion/ion reactions.^[25] Alternately pulsed nESI emitters allowed for sequential ion injection into the q2 reaction cell.^{[26, 27]} Doubly deprotonated reagent anions were ionized first and accumulated in the q2 reaction cell. Next, peptide cations were generated and transferred to the q2 cell to undergo a mutual storage reaction for 500–1000 milliseconds. The product ions were transferred to the Q3 ion trap, where subsequent MSⁿ analyses and mass analysis via mass-selective axial ejection (MSAE) were performed.^[28]

RESULTS AND DISCUSSION

Angiotensin II

The species that was first noted to give rise to an adducted CID product ion that was inconsistent with Schiff base formation, angiotensin II, is described here to illustrate the phenomenology and to compare its behavior when reacted with dianions of BDSA. The ion trap CID of FBDSA- and BDSA-modified angiotensin II (i.e., [M+♦]⁻ and [M+BDSA-H]⁻) are shown in Figure 1. In the ion/ion reaction of angiotensin II [M+H]⁺ with doubly deprotonated FBDSA, a negatively charged complex comprised of the two reactants (i.e., [M+FBDSA-H]⁻) is generated. When this complex is isolated and subjected to CID, an abundant water loss product is formed. The water loss product is represented as [M+♦]⁻, which is consistent with the dehydration reaction that leads to Schiff base formation. Water loss, however, is a very common process with peptide ions and does not, in itself, signal the covalent reaction. Subsequent collisional activation of the dehydration product generates several product ions with mass shifts consistent with the covalent Schiff base formation or electrostatic modification. These ions are labeled with the diamond symbol (♦). Ion trap CID of the [M+♦]⁻ ion produces several b♦-ions (the negative fragment ion nomenclature used here is adapted from protonated peptide literature^[29]) and two y₇-related ions (viz., y₇♦⁻ and y₇+FBDSA⁻) (Figure 1 (a)). The presence of b♦-ions in the product spectrum is consistent with the covalent modification of the N-terminus, which is the only primary amine site in angiotensin II. The greater contributions of the b₁♦- and b₆♦-ions compared to the other b♦-ions can be attributed to the well-established C-terminal cleavage at aspartate and N-terminal cleavage at the proline residue.^{[30, 31]} The b₁♦-ion is particularly noteworthy in that b₁-ions are rarely observed in the CID of unmodified peptide cations or anions.^{[32, 33]} The presence of the y₇♦-ion is inconsistent with the interpretation that Schiff base formation takes place at the N-terminus, at least for the fraction of precursor ions that fragment to give this product ion. This observation led to the hypothesis that some of the [M+♦]⁻ ions were comprised of attached FBDSA with water loss arising from elsewhere in the peptide. This hypothesis requires that the initial [M+FBDSA]⁻ complex can lose water while retaining an electrostatically bound FBDSA reagent and that subsequent activation of the electrostatically bound [M+♦]⁻ species can fragment along the peptide backbone while retaining the adduct. Evidence that both can occur is provided by the product ion that is consistent with the y₇+FBDSA assignment (see Figure 1(a)).

Figure 1.

Ion trap CID product ion spectra of a) FBDSA-modified product, [M+♦]⁻, b) BDSA-modified product, [M+BDSA-H]⁻, M = angiotensin II

While the above observations are supportive of the hypothesis described above, it is possible that, for example, arginine might also undergo Schiff base formation, which could explain the presence of the y₇♦-ion, and the signal assigned as y₇+FBDSA may have a different origin. The control experiment with [BDSA-2H]²⁻ as the reagent, which cannot engage in Schiff base formation, provides further support for the hypothesis. The reaction of [BDSA-2H]²⁻ with singly protonated angiotensin II produces a negatively charged, long-lived complex, [M+BDSA-H]⁻. Ion trap CID of [M+BDSA-H]⁻ is shown in Figure 1(b). Fragments with an associated double dagger symbol (ǂ) are consistent with the mass addition of [BDSA-2H]⁻. The major contribution to the fragmentation spectrum is the generation of the y₇ǂ-ion, which supports the y₇+FBDSA assignment made in Figure 1(a) The dehydration product in Figure 1(b) (i.e., the [M+BDSA-H-H₂O]⁻ ion) was isolated and subjected to ion trap CID resulting in formation of both y₇ǂ and y₇ǂ-H₂O products (see Supporting Information Figure S-1). The appearance of the y₇ǂ-H₂O-ion confirms that dehydration can occur within the y₇-ion and fully supports the hypothesis for the origin of the y₇♦-ion in Figure 1(a). The presence of weak signals that correspond to bǂ-ions can be attributed to an interaction of the BDSA with the protonated arginine, which is the second residue of angiotensin II, with cleavage C-terminal to the attachment site. It is noteworthy that no evidence for b₁ǂ-ion formation is apparent in Figure 1(b) while the b₁♦-ion is prominent in Figure 1(a). Covalent modification of the N-terminus in solution is a strategy that has been used to enhance b₁-ion formation in peptide tandem mass spectra.^34,35,36 It is also noteworthy that the [BDSA-H]⁻ signal following CID, which arises from detachment of the reagent from the complex as a singly charged species due to proton transfer, is quite small and essentially no evidence for loss of neutral BDSA is observed. This confirms that cleavage of covalent bonds can be highly competitive with loss of the non-covalently bound BDSA^{[37, 38, 39]}.

The angiotensin II data support the hypothesis that the [M+♦]⁻ species formed from the dehydration of the [angiotensin II+FBDSA]⁻ adduct is comprised of a mixture of structures. While the nominal structure of the Schiff base product is clear (i.e. Schiff base formation at the N-terminus) (see Figure 2(a)), several possibilities exist for the nature of the electrostatic interaction. One possibility involves the interaction of the charged guanidinium group of arginine and the anionic sulfonate group on FBDSA (i.e., a strong acid-base interaction), which is illustrated in Figure 2(b). Another possible non-covalent interaction could be the negatively charged proton bound dimer between a carboxylate group of the peptide and a sulfonate group of FBDSA (i.e., ⁻O₃S-C₇H₅O-SO₃⁻--H⁺--⁻OOC-peptide). A third possibility that is open to disulfonic acid reagents is a combination of a strong acid-base interaction involving one of the sulfonate groups with the other sulfonate group being engaged in proton sharing with a carboxylate group (i.e., a combination of the two interactions mentioned above). A fourth possibility is a salt-bridge structure in which sulfonate and carboxylate groups interact with a protonation site, depicted here as HO₃S-C₇H₅O-SO₃ ⁻--RH⁺--⁻OOC-peptide, where R represents the arginine side-chain. Neither the abundant y₇♦- and yǂ-ions nor the bǂ-ions in the spectra of Figure 1 can be used to distinguish between the four possibilities because of the aspartic acid residue at the N-terminus and the carboxylate group of the C-terminus. Therefore, experiments were conducted with protonated methyl esterified angiotensin II (i.e., both the aspartic acid sidechain and the C-terminus were methyl esterified) in reaction with [BDSA-2H]²⁻. This experiment serves to eliminate the possibilities for the three electrostatic interactions involving carboxylate groups. Ion trap CID of the [D(OMe)RVYIHPF(OMe)+BDSA-H]⁻ species generated a spectrum (Figure S-2) with methanol loss being the major process while the generation of [BDSA-H]⁻ and water loss were observed at lower levels. Very little backbone cleavage was noted, which is in stark contrast to the data for the unmodified [DRVYIHPF+BDSA-H]⁻ ion (Figure 1(b)). This result clearly indicates that at least one of the carboxyl groups of angiotensin II plays an important role in stabilizing the BDSA adduct so that backbone bond cleavage can compete with loss of BDSA.

Figure 2.

Ion population of [M+♦]⁻, a) Schiff base formation with singly protonated angiotensin II and doubly deprotonated FBDSA, b) One of the possible strong electrostatic interactions that may play a role in the observation of the y₇♦-ion of Figure 1(a). This interaction involves a deprotonated sulfonate group on FBDSA and the charged guanidinium side chain of arginine.

Leucine enkephalin-arginine

A series of model peptides based on leucine enkephalin were examined to provide further insights regarding the nature of the electrostatic binding of FBDSA and BDSA reagent anions to peptide cations. Figure 3 illustrates the four examples for electrostatic binding mentioned above for the YGGFLR case. The model peptide YGGFLR was subjected to charge inversion ion/ion reactions with doubly deprotonated anions derived either from FBDSA or BDSA and then activated via ion trap CID (Figures 4(a) and 4(b), respectively). The fragmentation behavior of the modified peptides was also compared to that of the unmodified deprotonated peptide, [M–H]⁻ (Figure S-3). This peptide was chosen so that the N-terminus and the arginine residues are at opposite ends of the sequence. In the cases of the modified versions of the peptide (i.e., [M+♦]⁻ and [M+BDSA]⁻) conditions were used to essentially deplete the precursor ion population completely (i.e., the activation amplitude was sufficiently high to dissociation essentially all of the precursor ion population) to avoid differential sampling of mixtures of structures, if present, that may have different kinetic stabilities. The [M+♦]⁻ ion was completely depleted and its location on the mass scale is indicated with an asterisk symbol (*) in Figure 4(a). Upon collisional activation of [M+♦]⁻, only N-terminally modified fragments are observed, which is consistent with covalent modification at the N-terminus. Many a♦- and b♦-ions along with neutral losses are observed in the product spectrum, which have been demonstrated previously to be common ion types generated via collisional activation of the [M+♦]⁻ species of other peptides.^{[17, 18]} In contrast with the angiotensin II data, no evidence for electrostatic binding in the [M+♦]⁻ ion is present in the sequence related product ions, such as y♦-ions. However, a relatively small signal consistent with [FBDSA-H]⁻ indicates that a small fraction of the precursor population may be electrostatically bound and fragments by loss of the neutral peptide, rather than cleavage of backbone bonds. Evidence for electrostatic binding and cleavage at backbone bonds has been observed, however, with arginine-containing tryptic peptides longer than approximately 8 or 9 residues.^[18]

Figure 3.

Four examples of possible non-covalent interactions between protonated YGGFLR and the dianion of BDSA. (a) Strong acid-base interaction. (b) Negatively charged proton-bound dimer. (c) Combined strong acid-base and proton-bound dimer interactions. (d) Salt-bridge interaction.

Figure 4.

Ion trap CID product ion spectra of a) FBDSA-modified product, [M+♦]⁻, b) BDSA-modified product, [M+BDSA-H]⁻, c) methyl esterified YGGFLR-BDSA adduct, [YGGFLR(OMe)+BDSA-H]⁻.

Charge inversion of YGGFLR via BDSA attachment (Figure 4(b)) illustrates a marked difference in fragmentation compared to the FBDSA modified peptide. Ion trap collisional activation of [M+BDSA-H]⁻ produces mainly yǂ-ions, which results from the interaction of the negatively charged reagent with the C-terminal arginine residue and/or the C-terminus. This observation differs drastically from the product spectrum of [M+♦]⁻, where the product ions are comprised solely of N-terminally modified fragment ions. The [M+BDSA-H]⁻ data provides further evidence that Schiff base modification is the dominant process via ion/ion reactions with the reagent FBDSA for YGGFLR. Charge inversion of YGGFLR via BDSA adduction also produced the most extensive C-terminal fragmentation in comparison to the other BDSA-modified peptides (Figure 4(b) and below). Ion trap CID of [M+BDSA-H]⁻ produces mainly yǂ-ions with a high relative abundance. Most notable is the high contribution of y₄ǂ, where the relative abundance is higher than that of the loss of [BDSA-H]⁻. In all other model peptides used (i.e., peptides not containing arginine), the most dominant dissociation pathway occurred via loss of negatively charged [BDSA-H]⁻ and the generation of the neutral peptide produced by proton transfer within the long lived complex, as shown in process (3):

$${[\mathrm{M}+\text{BDSA}-\mathrm{H}]}^{-}\to \mathrm{M}+{[\text{BDSA}-\mathrm{H}]}^{-}$$ (3)

This observation indicates that the electrostatic interaction between YGGFLR and the reagent is relatively strong and can compete with the dissociation of covalent bonds upon collisional activation. Figure 4(c) shows the ion trap CID product ion spectrum of the methyl esterified version of YGGFLR subjected to charge inversion with BDSA (i.e. CID of [YGGFLR(OMe)+BDSA-H]⁻. In this case, process (3) is dominant. Hence, consistent with the methyl esterification experiment with angiotensin II described above, it is apparent that the carboxyl group of the C-terminus is involved in the strong electrostatic binding that allows backbone cleavage to compete with formation of [BDSA-H]⁻ in the YGGFLR experiment.

The dissociation behaviors apparent in Figures 4(a) and 4(b) are clearly distinct from one another and illustrate the different ways in which covalent and electrostatic adduction can alter dissociation patterns. The ion trap CID of the unmodified peptide anion fragments quite distinctly from either of the modified forms of the peptide (see Figure S-3), as seen previously with other peptide sequences.^{[17, 18]} Ion trap CID of deprotonated YGGFLR produces significantly different relative contributions of the c₃- and b₅-ions, as well as the peaks associated with losses from the arginine side chain, such as loss of HN=C=NH. Ion trap CID of the anions derived from YGGFLH, YGGFLK, YGGFLF, and YGGFL exhibit the similar marked differences compared to the spectra of the respective modified species and are also included as Supplemental Information (Figures S-4,S-5,S-6,S-7(c), respectively).

Leucine enkephalin-histidine/lysine

Other model peptides, such as YGGFLH and YGGFLK, were subjected to ion/ion reactions to examine the CID behavior of protonated peptides without arginine but with other basic residues (see Figures 5 and 6, respectively). Ion trap CID of [YGGFLH+♦]⁻ (Figure 5(a)) produces similar dissociation behavior as YGGFLR (Figure 4(a)). The major ion-types observed are N-terminally modified fragments, such as a♦- and b♦-ions, which is consistent with covalent modification of the N-terminus. The relatively small signal consistent with [FBDSA-H]⁻ is the only indication of electrostatic binding in the [YGGFLH+♦]⁻ ion population. Ion trap CID of [YGGFLK+♦]⁻, on the other hand, exhibits distinct dissociation behavior from all other model peptides due to the major contribution of the y♦-ions, especially the contribution of the y₄♦-ion (Figure 6(a)). Collisional dissociation of [YGGFLK+♦]⁻ also produces a b₅♦-ion of low abundance. The peptide YGGFLK contains two primary amines, where one is located at the N-terminus and the other is present at the C-terminal lysine, so the presence of b♦- and y♦-ions can both be consistent with Schiff base formation. The preponderance of y♦-ions suggests that most of the modification in this case takes place at the lysine residue. A relatively small signal consistent with [FBDSA-H]⁻ is also observed in the [YGGFLK+♦]⁻ spectrum, which is the only clear indication that some of the [YGGFLK+♦]⁻ ions show electrostatic binding.

Figure 5.

Ion trap CID product ion spectra of a) FBDSA-modified product, [M+♦]⁻, b) BDSA-modified product, [M+BDSA-H]⁻ derived from M = YGGFLH

Figure 6.

Ion trap CID product ion spectra of a) FBDSA-modified product, [M+♦]⁻, b) BDSA-modified product, [M+BDSA-H]⁻ derived from M = YGGFLK

The dissociation behavior of BDSA-modified YGGFLH and YGGFLK (Figures 5(b) and 6(b), respectively) exhibit similar characteristics and are also distinct from their respective [M+♦]⁻ product ion spectra. The major fragmentation channel following collisional activation of BDSA-modified YGGFLH and YGGFLK (i.e., [M+BDSA-H]⁻) is the loss of the neutral peptide and generation of [BDSA-H]⁻. Smaller contributions of bǂ- and yǂ-ions are noted in both cases. The major sequence-related product ion in both cases is the y₄ǂ-ion. The total signal associated with yǂ-ions is much greater than that for bǂ-ions, which suggests that the electrostatic binding is largely at the C-terminal basic residue. However, the fact that any bǂ- ions are noted at all indicates electrostatic binding at the N-terminus for at least some of the ions. This dissociation behavior of BDSA-modified YGGFLH and YGGFLK is qualitatively similar to that of BDSA-modified YGGFLR in that they all show some backbone cleavage as well as loss of neutral peptide to give deprotonated BDSA. The relative contributions for backbone cleavages follows the order R>H>K, which likely reflects the relative strengths of the sulfonate-charge site interactions for these protonated side chains.

Leucine enkephalin-phenylalanine and leucine enkephalin

The model peptides YGGFLF and YGGFL were subjected to ion/ion reactions to investigate the behavior of peptide ions without a basic amino acid residue. Ion trap CID of FBDSA-modified YGGFLF, (i.e., [M+♦]⁻) exhibits dissociation behavior similar to the corresponding ions of YGGFLR and YGGFLH in that the product ion spectra are dominated by N-terminal fragments (see Figure 7(a)). Similar cleavages producing a♦- and b♦-ions are observed as well as similar relative contributions, where major contributions from the b₅♦- and b₄♦-ions are observed. The results of of YGGFL (Figure S-7(a)) are highly analogous to those of YGGFLF. The CID product spectrum of [YGGFL+♦]⁻ produces all N-terminal fragments, which is consistent with the expectation that the covalent modification takes place at the N-terminus. The only evidence for a contribution from ions with an electrostatic interaction comes from a minor signal consistent with [FBDSA-H]⁻.

Figure 7.

Ion trap CID product ion spectra of a) FBDSA-modified product, [M+♦]⁻, b) BDSA-modified product, [M+BDSA-H]⁻, derived from M = YGGFLF

Collisional activation of the electrostatic complex (i.e., [M+BDSA-H]⁻) for YGGFLF (Figure 7(b)) leads to similar dissociation behavior as that noted for the analogous species from YGGFLH and YGGFLK. Loss of [BDSA-H]⁻ again is the dominant dissociation pathway generated via ion trap CID of BDSA-modified YGGFLF and the major backbone cleavage yields the y₄ǂ-ion. The results from the [YGGFLF+H]⁺/[BDSA-2H]²⁻ experiment are also highly illustrative of the [YGGFL+H]⁺/[BDSA-2H]²⁻ experiment (Figure S-7(b)). Ion trap CID of BDSA-modified YGGFL predominantly leads to [BDSA-H]⁻ and the most abundant backbone fragment arises from cleavage of the analogous peptide linkage, which leads to a y₃ǂ-ion for this shorter peptide. Data were also collected for cations of N-terminally acetylated YGGFL, methyl-esterified YGGFL, and YGGFL with both N-terminal acetylation and C-terminal methylesterification, in reaction with doubly deprotonated [BDSA-2H]²⁻. In all cases, [M+BDSA-H]⁻ ions were formed and, in all cases, ion trap collisional activation led to strongly dominant [BDSA-H]⁻ formation (see Figure S-8). Collectively, the experiments with the YGGFLX model ions clearly show that strong electrostatic binding involving the BDSA anions takes place with peptide cations and that collisional activation of the electrostatically-bound cations can give rise to competitive backbone cleavage. However, backbone cleavage is most strongly competitive, by far, when arginine is present and at least one carboxyl group are both present in the peptide. In all other cases, loss of the peptide and formation of the BDSA anion is the most favored process. The dissociation behavior of BDSA-modified methyl esterified model peptides (i.e., YGGFLR-OMe and YGGFL-OMe) demonstrates the increased strength of the electrostatic interaction between the carboxylate groups and the reagent anion. This set of observations points to the importance of the interactions that involve both the protonated site of the peptide and a carboxyl group of the peptide (i.e., structures (c) and (d) of Figure 3).

CONCLUSIONS

These studies have added new insights into the nature of the non-covalent interactions of benzene disulfonic acid anions with peptide cations. The substitution of two sulfonic acid groups on the ring of benzaldehyde (i.e., FBDSA) has proved to be important in the observation of gas-phase Schiff base formation involving primary amines in peptide ions. These studies have confirmed earlier speculation that non-covalent interactions in the peptide-BDSA/FBDSA complex can be sufficiently strong to allow covalent bond cleavage to compete with loss of BDSA/FBDSA as an anion or neutral. Such an interpretation was forthcoming from the observation of adduct-containing fragments that did not contain a primary amine for Schiff base formation. Studies with model protonated YGGFLX peptides clearly show that two criteria greatly facilitate the observation of covalent bond cleavage versus disruption of non-covalent binding. These include the presence of a protonated arginine, histidine or lysine, with arginine giving rise to the strongest acid-base interaction of all of the basic side chains, and the presence of a carboxyl group, which can engage in proton sharing with a sulfonate group or stabilize a salt-bridge interaction. While the data presented here cannot be used to distinguish the latter two possibilities, our experience with benzene sulfonic acid (i.e., a single sulfonate group present on the reagent anion) in reaction with multiply protonated peptides (data not shown), which should be able to undergo a salt-bridge type interaction, show dominant loss of the reagent from complexes with the peptide. This leads us to favor the combined acid-base/proton-bound dimer interaction involving both sulfonate groups (i.e., the interaction of structure (c) in Figure 3).

The improved understanding derived from this work regarding the nature of non-covalent interactions in ion/ion chemistry is useful in considering the design of reagents for specific purposes. For example, the ‘tuning’ of binding strength between reagent and analyte ion via control over the identities of functional groups in both the analyte and reagent can be used to either enhance or minimize particular reaction channel types. For example, it may be possible to increase the cleavage of covalent bonds in ion/ion adducts by incorporating an additional sulfonate group in the reagent. It might also be possible to minimize covalent bond cleavage in arginine containing peptides by using a reagent dianion with one sulfonate group and one carboxylate group instead of two sulfonate groups. This work has indicated, for example, that contributions from covalent bond cleavage from electrostatically bound adducts can be minimized by esterification of the peptide carboxyl groups. The results reported here, therefore, provide useful new insights in the design and application of reagents for gas phase ion/ion reactions.