Ion-Ion and Ion-Molecule Reactions at the Surface of Proteins Produced by Nanospray. Information on the Number of Acidic Residues and Control of the Number of Ionized Acidic and Basic Residues

Mass Spectra of charge states of folded proteins were obtained with nanospray and aqueous solution containing 20 μM the protein (ubiquitin, cytochrome c, lysozyme) and one of the NaA salts NaCl, NaI, NaAc (acetate) (1–10 mM). At very low collision activated decomposition (CAD), the mass spectra of a protein with charge z exhibited a replacement of zH+ with zNa+ and also multiple adducts of NaA. Higher CAD converts the NaA adduct peaks to Na minus H peaks. These must be due to loss of HA where the H was provided by the protein. The degree of HA loss with increasing CAD followed the order I < Cl < Ac. Significantly, the intensity of the ions with n (Na minus H) adducts showed a downward break past an nMAX which is equal to the number of acidic residues of the protein plus the charge of the protein. All the observations could be rationalized within the framework of the electrospray mechanism and the charge residue model, which predict that due to extensive evaporation of solvent, the solutes will reach very high concentrations in the final charged droplets. At such high concentrations, positive ions such as Na+, NH4+ form ion pairs with ionized acidic residues and the negative A− form ion pairs with ionized basic residues of the protein. Adducts of Na+, and NaA to backbone amide groups occur also. This reaction mechanism fits all the experimental observations and provides predictions that the number of acidic and basic groups at the surface of the gaseous protein that remain ionized can be controlled by the absence or presence of additives to the solution

Infrared Multiple-Photon Dissociation Spectroscopy of Tripositive Ions: Lanthanum–Tryptophan Complexes

Collision-induced charge disproportionation limits the stability of triply charged metal ion complexes and has thus far prevented successful acquisition of their gas-phase IR spectra. This has curtailed our understanding of the structures of triply charged metal complexes in the gas phase and in biological environments. Herein we report the first gas-phase IR spectra of triply charged La^III complexes with a derivative of tryptophan (<i>N</i>-acetyl tryptophan methyl ester), and an unusual dissociation product, a lanthanum amidate. These spectra are compared with those predicted using density functional theory. The best structures are those of the lowest energies that differ by details in the π-interaction between La³⁺ and the indole rings. Other binding sites on the tryptophan derivative are the carbonyl oxygens. In the lanthanum amidate, La³⁺ replaces an H⁺ in the amide bond of the tryptophan derivative

Metal Ion Complexes with HisGly: Comparison with PhePhe and PheGly

Gas-phase complexes of five metal ions with the dipeptide HisGly have been characterized by DFT computations and by infrared multiple photon dissociation spectroscopy (IRMPD) using the free electron laser FELIX. Fine agreement is found in all five cases between the predicted IR spectral features of the lowest energy structures and the observed IRMPD spectra in the diagnostic region 1500–1800 cm^–1, and the agreement is largely satisfactory at longer wavelengths from 1000 to 1500 cm^–1. Weak-binding metal ions (K⁺, Ba²⁺, and Ca²⁺) predominantly adopt the charge-solvated (CS) mode of chelation involving both carbonyl oxygens, an imidazole nitrogen of the histidine side chain, and possibly the amino nitrogen. Complexes with Mg²⁺ and Ni²⁺ are found to adopt iminol (Im) binding, involving the deprotonated amide nitrogen, with tetradentate chelation. This tetradentate coordination of Ni­(II) is the preferred binding mode in the gas phase, against the expectation under condensed-phase conditions that such binding would be sterically unfavorable and overshadowed by other outcomes such as metal ion hydration and formation of dimeric complexes. The HisGly results are compared with corresponding results for the PheAla, PheGly, and PhePhe ligands, and parallel behavior is seen for the dipeptides with N-terminal Phe versus His residues. An exception is the different chelation pattern determined for PhePhe versus HisGly, reflecting the intercalation-type cation binding pocket of the PhePhe ligand. The complexes group into three well-defined spectroscopic patterns: nickel and magnesium, calcium and barium, and potassium. Factors leading to differentiation of these distinct spectroscopic categories are (1) differing propensities for choosing the iminol binding pattern, and (2) single versus double charge on the metal center. Nickel and magnesium ions show similar gas-phase binding behavior, contrasting with their quite different patterns of peptide interaction in condensed phases

a₂ Ion Derived from Triglycine: An N₁-Protonated 4-Imidazolidinone

Fragmentation of protonated peptides in the gas phase constitutes the basis for gas-phase sequencing of peptides using tandem mass spectrometry. Several mechanistic studies have indicated possible loss of b_<i>n</i> ion sequence information as a consequence of macrocycle formation from internal nucleophilic attacks. Here, we show by infrared multiple-photon dissociation spectroscopy and density functional theory that the prototypical a₂ ion generated from protonated triglycine is predominantly a cyclic N₁-protonated 4-imidazolidinone. Cyclization resulting from internal nucleophilic attacks therefore may be a more general phenomenon than anticipated