Spectroscopic Evidence for Peptide-Bond-Selective Ultraviolet Photodissociation.

We study the photodissociation induced by ultraviolet excitation of amide bonds in gas-phase protonated peptides. Jointly, mass spectrometry and cold ion spectroscopy provide evidence for a selective nonstatistical dissociation of specific peptide bonds in the spectral region of the formally forbidden n → π* transition of amide groups. Structural analysis reveals that the activation of this transition, peaked at 226 nm, originates from the nonplanar geometry of the bond. In contrast, the statistical dissociation in the electronic ground state appears to be the main outcome of the π → π* excitation of the peptide bonds at 193 nm. We propose a tentative model that explains the difference in the fragmentation mechanisms by the difference in localization of the electronic transitions and the higher amount of vibrational energy released in the electronic excited state upon absorption at 193 nm.

Gas-phase structures reflect the pain-relief potency of enkephalin peptides.

We use cold ion spectroscopy and quantum-chemical computations to solve the structures of opioid peptides enkephalins in the gas phase. The derived structural parameters clearly correlate with the known pharmacological efficiency of the studied drugs, suggesting that gas-phase methods, perhaps, can be used for predicting the relative potency of ligand drugs that target the hydrophobic pockets of receptors.

Method for Identification of Threonine Isoforms in Peptides by Ultraviolet Photofragmentation of Cold Ions.

Identification of isomeric amino acid residues in peptides and proteins is challenging but often highly desired in proteomics. One of the practically important cases that require isomeric assignments is that associated with single-nucleotide polymorphism substitutions of Met residues by Thr in cancer-related proteins. These genetically encoded substitutions can yet be confused with the chemical modifications, arising from protein alkylation by iodoacetamide, which is commonly used in the standard procedure of sample preparation for proteomic analysis. Similar to the genetically encoded mutations, the alkylation also induces a conversion of methionine residues, but to the iso-threonine form. Recognition of the mutations therefore requires isoform-sensitive detection techniques. Herein, we demonstrate an analytical method for reliable identification of isoforms of threonine residues in tryptic peptides. It is based on ultraviolet photodissociation mass spectrometry of cryogenically cooled ions and a machine-learning algorithm. The measured photodissociation mass spectra exhibit isoform-specific patterns, which are independent of the residues adjacent to threonine or iso-threonine in a peptide sequence. A comprehensive metric-based evaluation demonstrates that, being calibrated with a set of model peptides, the method allows for isomeric identification of threonine residues in peptides of arbitrary sequence.

Peptide Bond Ultraviolet Absorption Enables Vibrational Cold-Ion Spectroscopy of Nonaromatic Peptides.

Peptide-bond VUV absorption is inherent to all proteins and peptides. Although widely exploited in top-down proteomics for photodissociation, this absorption has never been spectroscopically characterized in the gas phase. We have measured VUV/UV photofragmentation spectrum of a single peptide bond in a cryogenically cold protonated dipeptide. Although the spectrum appears to be very broadband and structureless, vibrational pre-excitation of this and even larger cold peptides significantly increases the UV dissociation yield for some of their photofragments. We use this effect to extend the technique of IR-UV photofragmentation vibrational spectroscopy, developed for aromatic peptides, to nonaromatic ones and demonstrate measurements of conformation-specific and nonspecific IR spectra for di- to hexa-peptides.