Determination of the Gas-phase Acidities of Oligopeptides

The acidities of amino acid residues are among the most important thermochemical properties that influence the structures, the reactivity, and the folding-unfolding processes of proteins ^9,19. Individual amino acid residues often show different effective acidities depending on their locations in proteins. In particular, the residues located at the active sites often exhibit significantly perturbed acidities. One such example is the cysteine residue residing in the active sites of the thioredoxin super-family of enzymes ^20,21. The active site cysteine is unusually acidic compared to those in unfolded proteins ^3-5. It has been suggested that the helical conformation may have a significant contribution to the unusual acidity. There are extensive experimental studies on the acid-base properties of peptides carried out in solutions, especially in aqueous solutions ^2,6-8. The results were often complicated by solvent effects ⁷. In fact, most of the active sites in proteins are located near the interior region where solvent effects are minimized ^9,10.

In order to understand intrinsic acid-base properties of peptides and proteins, it is important to carry out the studies in a solvent-free environment. Here we introduce a mass spectrometry-based method for the determination of the gas-phase acidity. The approach is referred to as the extended Cooks kinetic method. This method has been successfully applied to a wide range of chemical systems for the determination of various thermochemical properties, such as the gas-phase acidity, the proton affinity, the metal ion affinity, the electron affinity, and the ionization energy ^11-15,22-26. We have applied this method to determine the gas-phase acidities of a series of oligo cysteine-polyalanine and cysteine-polyglycine peptides ^17,18,27. These studies show that the N-terminal cysteine peptides are significantly more acidic than the corresponding C-terminal ones. The high acidities of the former are likely due to the helical conformational effects in which the thiolate anion is strongly stabilized by the interaction with the helix macro-dipole. Because of the non-volatile and thermally labile nature of peptides, the kinetic method is the most practical approach available at present to produce reasonably accurate acid-base thermochemical quantities of peptides ²⁸.

The general scheme and the equation associated with the kinetic method are shown in Figure 1. The determination of the gas-phase acidity of a peptide (AH) starts with the formation of a series of proton-bound cluster anions, [A•H•A_i]¯ (or [A¯•H⁺•A_i¯]¯), in the ion source region of the mass spectrometer, where A¯ and A_i¯ are the deprotonated forms of the peptide and the reference acids, respectively. The reference acids are organic compounds with known gas-phase acidities. The reference acids should have structures similar to each other (but not necessarily similar to that of the peptide). The similarity of the structures between reference acids ensures the similarity of the entropies of deprotonation among them. The proton-bound cluster anions are mass selected and collisionally activated and subsequently dissociated using collision-induced dissociation (CID) experiments to yield the corresponding monomeric anions, A¯ and A_i¯, with rate constants of k and k_i, respectively, shown in Figure 1a. If secondary fragmentations are negligible, the abundance ratio of the CID fragment ions, [A¯]/[A_i¯], represents an approximate measure of the ratio of the rate constants, k/k_i. Under the assumption that there are no reverse activation barriers for both dissociation channels, the CID product ion branching ratios, ln[A¯]/[A_i¯], will be linearly correlated to the gas-phase acidity of the peptide (Δ_acidH) and those of the reference acids (Δ_acidH_i), as shown in Figure 1b. In this equation, Δ_acidH_avg is the average gas-phase acidity of the reference acids, Δ(ΔS) is the entropy term (which can be assumed constant if the reference acids are structurally similar to each other), R is the universal gas constant, and T_eff is the effective temperature of the system. The effective temperature is an empirical parameter that depends on several experimental variables, including the collision energy.

The value of the gas-phase acidity is determined by constructing two sets of thermo-kinetic plots. The first set is obtained by plotting ln([A¯]/[A_i¯]) against Δ_acidH_i – Δ_acidH_avg, as shown in Figure 4a. Linear regression will yield a set of straight lines with the slopes of X = 1/RT_eff and intercepts of Y = – [Δ_acidH – Δ_acidH_avg]/RT_eff – Δ(ΔS)/R. The second set of plots is obtained by plotting the resulting intercepts (Y) from the first set against the corresponding slopes (X), as shown in Figure 4b. Linear regression produces a new line with a slope of Δ_acidH – Δ_acidH_avg and an intercept of Δ(ΔS)/R. The value of Δ_acidH is then obtained from the slope and the entropy term, Δ(ΔS), is obtained from the intercept.

The experiments are performed using a triple quadrupole mass spectrometer interfaced to an electrospray ionization (ESI) ion source. A schematic diagram of the mass spectrometer is shown in Figure 2. The CID experiments are performed by mass selecting the proton-bound cluster anions with the first quadrupole unit and allowing them to undergo collisions with argon atoms leaked into the collision chamber which is held at a pressure of around 0.5 mTorr. The dissociation product ions are mass analyzed with the third quadrupole unit. The CID spectra are recorded at several collision energies with the m/z range wide enough to cover all possible secondary fragments. The CID product ion intensities are measured by setting the instrument in the selected reaction monitoring (SRM) mode in which the scan is focused on selected product ions. The CID experiments are performed at four different collision energies, corresponding to center-of-mass energies (E_cm) of 1.0, 1.5, 2.0, and 2.5 eV, respectively. The center-of-mass energy is calculated using the equation: E_cm = E_lab [m/(M+m)], where E_lab is the collision energy in the laboratory frame, m is the mass of argon, and M is the mass of the proton-bound cluster ion.

In this article, we use the oligopeptide Ala₃CysNH₂ (A₃CH) as the model compound. The C-terminus is amidated and the thiol group (SH) of the cysteine residue will be the acidic site. The selection of the suitable reference acids is crucial for the successful measurement of the gas-phase acidity. The ideal reference acids are structurally similar (to each other) organic compounds with well-established gas-phase acidity values. The reference acids should have acidity values close to that of the peptides. For the peptide A₃CH, six halogenated carboxylic acids are chosen as the reference acids. The six reference acids are chloroacetic acid (MCAH), bromoacetic acid (MBAH), difluoroacetic acid (DFAH), dichloroacetic acid (DCAH), dibromoacetic acid (DBAH), and trifluoroacetic (TFAH). Two of them, DFAH and MBAH, will be used to illustrate the protocol.