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Gas-phase acidities (GA or Δ G acid ) of acidic di- and tripeptides are determined for the first time. The peptides studied are composed of inert alanine (A) residues and one X residue of either aspartic acid (D) or glutamic acid (E): AX, XA, AAX, AXA, and XAA. Experimental GAs were measured by the thermokinetic method of deprotonation ion/molecule reactions in a Fourier transform ion cyclotron resonance mass spectrometer. Calculated GAs were obtained by composite correlated molecular orbital theory at the G3(MP2) level for deprotonation of carboxylic acid groups both at the C-terminus and at the side chain. Excellent agreement was found between experimental and calculated GA values. There is a slight preference for peptides with D being more acidic than analogous peptides with E, which agrees with the GAs of the corresponding amino acids. Experiments showed that peptides are more acidic (lower numerical GA values) when the acidic residue is located at the C-terminus (i.e., AX or AAX). The lowest energy form of deprotonated AAE has a unique structure where the longer side chain of E allows the two carboxylates, which are in close proximity, to share the proton. The tripeptides are less acidic (higher GA value) by 3-7 kcal/mol when the acidic residue is in the center. The tripeptides are more acidic (by 2-10 kcal/mol) than dipeptides containing the same acidic residue at the same location.

Gas-phase acidities and heats of formation have been predicted at the G3(MP2)/SCRF-COSMO level of theory for 10 phosphorylated amino acids and their corresponding amides, including phospho-serine (pSer), -threonine (pThr), and -tyrosine (pTyr), providing the first reliable set of these values. The gas-phase acidities (GAs) of the three named phosphorylated amino acids and their amides have been determined using proton transfer reactions in a Fourier transform ion cyclotron mass spectrometer. Excellent agreement was found between the experimental and predicted GAs. The phosphate group is the deprotonation site for pSer and pThr and deprotonation from the carboxylic acid generated the lowest energy anion for pTyr. The infrared spectra were calculated for six low energy anions of pSer, pThr, and pTyr. For deprotonated pSer and pThr, good agreement is found between the experimental IRMPD spectra and the calculated spectra for our lowest energy anion structure. For pTyr, the IR spectra for a higher energy phosphate deprotonated structure is in good agreement with experiment. Additional experiments tested electrospray ionization (ESI) conditions for pTyr and determined that variations in solvent, temperature, and voltage can result in a different experimental GA value, indicating that ESI conditions affect the conformation of the pTyr anion.

Using proton-transfer reactions in a Fourier transform ion cyclotron resonance mass spectrometer and correlated molecular orbital theory at the G3(MP2) level, gas-phase acidities (GAs) and the associated structures for amides corresponding to the common amino acids have been determined for the first time. These values are important because amino acid amides are models for residues in peptides and proteins. For compounds whose most acidic site is the C-terminal amide nitrogen, two ions populations were observed experimentally with GAs that differ by 4-7 kcal/mol. The lower energy, more acidic structure accounts for the majority of the ions formed by electrospray ionization. G3(MP2) calculations predict that the lowest energy anionic conformer has a cis-like orientation of the [-C(═O)NH](-) group whereas the higher energy, less acidic conformer has a trans-like orientation of this group. These two distinct conformers were predicted for compounds with aliphatic, amide, basic, hydroxyl, and thioether side chains. For the most acidic amino acid amides (tyrosine, cysteine, tryptophan, histidine, aspartic acid, and glutamic acid amides) only one conformer was observed experimentally, and its experimental GA correlates with the theoretical GA related to side chain deprotonation.

Using mass spectrometry and correlated molecular orbital theory, three deprotonated structures were revealed for the amino acid tyrosine. The structures were distinguished experimentally by ion/molecule reactions involving proton transfer and trimethylsilyl azide. Gas-phase acidities from proton transfer reactions and from G3(MP2) calculations generally agree well. The lowest energy structure, which was only observed experimentally using electrospray ionization from aprotic solvents, is deprotonated at the carboxylic acid group and is predicted to be highly folded. A second unfolded carboxylate structure is several kcal/mol higher in energy and primarily forms from protic solvents. Protic solvents also yield a structure deprotonated at the phenolic side chain, which experiments find to be intermediate in energy to the two carboxylate forms. G3(MP2) calculations indicate that the three structures differ in energy by only 2.5 kcal/mol, yet they are readily distinguished experimentally. Structural abundance ratios are dependent upon experimental conditions, including the solvent and accumulation time of ions in a hexapole. Under some conditions, carboxylate ions may convert to phenolate ions. For phenylalanine, which lacks a phenolic group, only one deprotonated structure was observed experimentally when electrosprayed from protic solvent. This agrees with G3(MP2) calculations that find the folded and unfolded carboxylate forms to differ by 0.3 kcal/mol.

The gas-phase acidities (GAs) of six tripeptides (GlyGlyGly, GlyAlaGly, AlaGlyAla, AlaAlaAla, AibAibAib, and SarSarSar) and their methyl esters were obtained by proton transfer reactions in a Fourier transform ion cyclotron resonance mass spectrometer and G3(MP2) molecular orbital theory calculations. All six peptides have GAs in the range 321.0-323.7 kcal/mol. Their deprotonation to produce [M - H](-) occurs at the C-terminal carboxylic acid group. The tripeptides are about 10 kcal/mol more acidic than the amino acids glycine (Gly) and alanine (Ala). This is consistent with the extensive hydrogen bonding that was found in the tripeptide structures. For the methyl esters, deprotonation occurs at the peptide backbone. G3(MP2) calculations show that the most energetically favored site of deprotonation is an amide nitrogen, with the central amide being generally preferred. Nitrogen deprotonation requires 10-20 kcal/mol less energy than deprotonation at a methylene carbon. Only three of the methyl esters (GlyGlyGly-OMe, GlyAlaGly-OMe, and AlaAlaAla-OMe) deprotonate experimentally by electrospray ionization. Experimental GAs for these esters are in the range of 336.7-338.1 kcal/mol, in excellent agreement with the calculated G3(MP2) values. Experimental GAs could not be obtained for the other three methyl esters (AlaGlyAla-OMe, AibAibAib-OMe, and SarSarSar-OMe) because they did not produce sufficient deprotonated molecular ions. Trisarcosine methyl ester, SarSarSar-OMe, cannot be deprotonated at a central amide nitrogen because methyl groups are present at these sites; consequently, it has a high G3(MP2) GA value (less acidic) of 350.6 kcal/mol for deprotonation at the N-terminal nitrogen. For AlaGlyAla-OMe and AibAibAib-OMe, calculations of van der Waals and solvent accessible surfaces reveal that methyl groups are blocking the amide nitrogen sites. Therefore, conformational and steric hindrance effects are limiting the ability of these peptide methyl esters to deprotonate in the mass spectrometer.