Determination of the Gas-phase Acidities of Oligopeptides

Summary

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Abstract

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus ^1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions ^6-8, the results are often complicated by solvent effects ⁷. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized ^9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.

We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala₃CysNH₂ (A₃CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) ^11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots ^17,18.

The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

Introduction

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 ....

Protocol

1. Preparation of the Sample Solutions

1. First prepare the stock solutions of the peptide and the six reference acids using a mixed solvent of methanol and water in a 1:1 volume ratio. The stock solutions should have a concentration of about 10^-3 M.
2. Weigh out 1 mg of the solid peptide sample, A₃CH, in a 1.5 ml Eppendorf tube and add 1.0 ml of mixed solvent of methanol and water, and mix using a vortex.
3. Weigh out 1 mg of difluoroacetic acid (DFAH) and add 1.0 ml of the mixed solvent of methanol and water, and mix using a vortex.
4. Use the same procedure to make the stock solutions for the other five reference ....

Results

1. The CID bracketing experiments provide information on the relative acidities of the peptide compared to the selected reference acids. Two representative CID spectra of the peptide (A₃CH) with two reference acids, DFAH and MBAH, are shown in Figure 3. In Figure 3a the ion abundance (peak height) of the peptide ion is weaker than that of DFA¯, and in Figure 3b, the ion abundance of the peptide ion is stronger than that of MBA¯. The two spectra suggest that the gas-phase acidi.......

Discussion

The successful measurement of the gas-phase acidity of a peptide largely relies on the selection of suitable reference acids. The ideal reference acids are structurally similar organic compounds with well-established gas-phase acidity values. The reference acids should have similar structures to each other. This will ensure a similar entropy of deprotonation for each of the reference acids in the set. The reference acids should have acidity values close to those of the peptides. For shorter cysteine-containing oligopepti.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Mass Spectrometer	Varian	1200 L and 320 L
Chloroacetic acid	Sigma-Aldrich	402923
Bromoacetic acid	Sigma-Aldrich	B56307
Difluoroacetic acid	Sigma-Aldrich	142859
Dichloroacetic acid	Sigma-Aldrich	D54702
Dibromoacetic acid	Sigma-Aldrich	242357
Trifluoroacetic acid	Sigma-Aldrich	T6508