Ionizing radiation induces cross-linking of two noncovalently bound collagen mimetic peptide triple helices in the absence of a molecular environment.

Cross-linking is a fundamental molecular process that is highly important for many applications, in particular, to tune the properties of collagen-based biomaterials. Chemical reagents, the action of enzymes or physical factors such as heat or radiation can facilitate collagen cross-linking. Ionizing radiation has the advantages of being fast, efficient and free from potentially toxic reagents. Collagen cross-linking by ionizing radiation is thought to occur via a water-mediated pathway. In the past, synthesized collagen mimetic peptides have proven to be of great value for understanding the influence of the amino acid sequence on the stability of tertiary (fibrous) as well as secondary (triple helical) structures of collagen. Cross-linking of synthetic collagen mimetic peptides is often used for modifying the properties of biomaterials. In this work, for the first time, we apply radiation-induced cross-linking to synthetic collagen mimetic peptides and present an experimental and theoretical study of peptide hexamers consisting of two noncovalently bound triple helices in the absence of a molecular environment, i.e. in the gas phase. Our results show that X-ray photoabsorption of the hydroxylated hexamer leads to ionization and cross-linking of the two triple helices: thus, we found evidence that cross-linking can be achieved by ionizing radiation, without the presence of any reagent or water. We propose a cross-linking mechanism involving the creation of two radicals on hydroxyproline side-chains and their recombination, ultimately leading to covalent bond formation between the triple helices.

UV-VUV Photofragmentation Spectroscopy of Isolated Neutral Fragile Macromolecules: A Proof-of-Principle Based on a Deprotonated Vancomycin-Peptide Noncovalent Complex.

The gas phase offers the possibility to analyze organic molecules by ultraviolet-vacuum ultraviolet (UV-VUV) spectroscopy without any solvent effect or limitation in terms of spectral range due to absorption by the solvent. Up to now, the size and chemical composition of neutral molecular systems under study have been limited by the use of vaporization methods based on thermal heating. Soft sources of gas-phase thermolabile molecular systems such as electrospray or matrix-assisted laser desorption ionization are appealing alternatives to heating-based techniques, but they lead to the production of ions. In such cases, UV-VUV action spectroscopy is then the method of choice to study the electronic structure and corresponding photodynamics of these gas-phase molecular ions. However, previous investigations have shown that the UV-VUV action spectrum of a given molecular ion depends on the charge state, which in many cases might be a caveat. Here, by means of synchrotron radiation coupled to mass spectrometry and through the test case of the glycopeptide antibiotic vancomycin noncovalently bound to a deprotonated small peptide, we show that the UV-VUV photofragmentation spectrum of neutral thermally fragile organic molecules can be obtained via charge-tagging action spectroscopy.

Chemical Processes Involving 18-Crown-6-Ether in Activated Noncovalent Complexes with Protonated Peptides.

These last decades, it has been widely assumed that 18-crown-6-ether (CE) plays a spectator role during the chemical processes occurring in isolated host-guest complexes between peptides or proteins and CE after activation in mass spectrometers. Our present experimental and theoretical results challenge this hypothesis by showing that CE can abstract a proton or a protonated molecule from protonated peptides after activation by collisions in argon or electron capture/transfer. Furthermore, thanks to comparison between experimental and calculated values of collision cross-sections, we demonstrate that CE can change binding site after electron transfer. We also propose detailed mechanisms for these processes.

Photoinduced Processes within Noncovalent Complexes Involved in Molecular Recognition.

Investigating the intrinsic properties of molecular complexes is crucial for understanding the influence of noncovalent interactions on fundamental chemical reactions. Moreover, specific molecular recognition between a ligand and its receptor is a highly important biological process, but little is known about the effects of ionizing radiation on ligand-receptor complexes. The processes triggered by VUV photoabsorption on isolated noncovalent complexes between the glycopeptide antibiotic vancomycin and a mimic of its receptor have been probed by means of mass spectrometry and synchrotron radiation. In the case of protonated species, the glycosidic bond of vancomycin was cleaved with low activation energy, regardless of the molecular environment. In sharp contrast, for deprotonated species, electron photodetachment from carboxylate groups only triggered CO2 loss, whereas the glycosidic bond remained intact. Importantly, the noncovalent complex was also found to survive VUV photoabsorption only when the native structure is conserved in the gas phase.

Multiple valence electron detachment following Auger decay of inner-shell vacancies in gas-phase DNA.

We have studied soft X-ray photoabsorption in the doubly deprotonated gas-phase oligonucleotide [dTGGGGT-2H]2-. The dominating decay mechanism of the X-ray induced inner shell vacancy was found to be Auger decay with detachment of at least three electrons, leading to charge reversal of the anionic precursor and the formation of positively charged photofragment ions. The same process is observed in heavy ion (12 MeV C4+) collisions with [dTGGGGT-2H]2- where inner shell vacancies are generated as well, but with smaller probability. Auger decay of a single K-vacancy in DNA, followed by detachment of three or more low energy electrons instead of a single high energy electron has profound implications for DNA damage and damage modelling. The production of three low kinetic energy electrons with short mean free path instead of one high kinetic energy electron with long mean free path implies that electron-induced DNA damage will be much more localized around the initial K-shell vacancy. The fragmentation channels, triggered by triple electron detachment Auger decay are predominantly related to protonated guanine base loss and even loss of protonated guanine dimers is tentatively observed. The fragmentation is not a consequence of the initial K-shell vacancy but purely due to multiple detachment of valence electrons, as a very similar positive ion fragmentation pattern is observed in femtosecond laser-induced dissociation experiments.

Mass Spectral Signatures of Complex Post-Translational Modifications in Proteins: A Proof-of-Principle Based on X-ray Irradiated Vancomycin.

Characterizing post-translational modifications (PTM) of proteins is of key relevance for the understanding of many biological processes, as these covalent modifications strongly influence or even determine protein function. Among the different analytical techniques available, mass spectrometry is attracting growing attention because recent instrumental and computational improvements have led to a massive rise of the number of PTM sites that can be identified and quantified. However, multiple PTM occurring at adjacent amino acid residues can lead to complex and dense chemical patterns that are a challenge to characterize. By means of X-ray synchrotron radiation coupled to mass spectrometry, and through the test-case of the glycopeptide antibiotic vancomycin, we show that such a pattern has a unique and robust signature in terms of photon energy and molecular environment. This highlights the potential of this technique in proteomics and its value as a tool to understand the biological roles of PTM.