The initial stages of protein unfolding may reflect the stability of the entire fold and can also reveal which parts of a protein can be perturbed, without restructuring the rest. In this work, we couple UVPD with activated ion mobility mass spectrometry to measure how three model proteins start to unfold. Ubiquitin, cytochrome c and myoglobin ions produced via nESI from salty solutions are subjected to UV irradiation pre-mobility separation; experiments are conducted with a range of source conditions which alter the conformation of the precursor ion as shown by the drift time profiles. For all three proteins, the compact structures result in less fragmentation than more extended structures which emerge following progressive in-source activation. Cleavage sites are found to differ between conformational ensembles, for example, for the dominant charge state of cytochrome c [M + 7H], cleavage at Phe10, Thr19 and Val20 was only observed in activating conditions whilst cleavage at Ala43 is dramatically enhanced. Mapping the photo-cleaved fragments onto crystallographic structures provides insight into the local structural changes that occur as protein unfolding progresses, which is coupled to global restructuring observed in the drift time profiles. Graphical Abstract.
Infrared multiple photon dissociation (IRMPD) spectroscopy allows for the derivation of the vibrational fingerprint of molecular ions under tandem mass spectrometry (MS/MS) conditions. It provides insight into the nature and localization of posttranslational modifications (PTMs) affecting single amino acids and peptides. IRMPD spectroscopy, which takes advantage of the high sensitivity and resolution of MS/MS, relies on a wavelength specific fragmentation process occurring on resonance with an IR active vibrational mode of the sampled species and is well suited to reveal the presence of a PTM and its impact in the molecular environment. IRMPD spectroscopy is clearly not a proteomics tool. It is rather a valuable source of information for fixed wavelength IRMPD exploited in dissociation protocols of peptides and proteins. Indeed, from the large variety of model PTM containing amino acids and peptides which have been characterized by IRMPD spectroscopy, specific signatures of PTMs such as phosphorylation or sulfonation can be derived. High throughput workflows relying on the selective fragmentation of modified peptides within a complex mixture have thus been proposed. Sequential fragmentations can be observed upon IR activation, which do not only give rise to rich fragmentation patterns but also overcome low mass cutoff limitations in ion trap mass analyzers. Laser-based vibrational spectroscopy of mass-selected ions holding various PTMs is an increasingly expanding field both in the variety of chemical issues coped with and in the technological advancements and implementations.
Low-energy collision induced dissociation (CID) of deprotonated L-cysteine S-sulfate, [cysS-SO 3 ],delivered in the gas phase by electrospray ionization, has been found to provide a means to form deprotonated L-cysteine sulfenic acid, a fleeting intermediate in biological media. The reaction mechanism underlying this process is the focus of the present contribution. At the same time other novel species are formed, which were not observed in previous experiments. To understand fragmentation pathways of [cysS-SO 3 ]-, reactive chemical dynamics simulations coupled with a novel algorithm for automatic determination of intermediates and transition state were performed. This approach has allowed the identification of the mechanisms involved and to explain the experimental fragmentation pathways. Chemical dynamics simulations have shown that a roaming-like mechanism can be at the origin of L-cysteine sulfenic acid.
Cisplatin [cis-diamminedichloroplatinum(II)] was the first platinum-based antineoplastic agent and is still a cornerstone for the treatment of various solid tumors. Reactive events responsible for cisplatin activity are unveiled here at the molecular level. Simple ligands (L) representing ubiquitous functional groups in the biological environment likely to be encountered by administered cisplatin have been allowed to react with cis-[PtCl(NH3)2 (H2O)](+), the primary intermediate from cisplatin hydrolysis. The substitution reactions have been examined by a combined experimental and computational approach and the structural features of the substitution product, cis-[PtCl(NH3)2(L)](+), have been probed by IR multiple-photon dissociation (IRMPD) spectroscopy. Furthermore, IRMPD spectroscopy has been exploited to elucidate the structure of [PtCl(NH3)2(L)(H2O)](+) clusters, also obtained by electrospray ionization (ESI) from the aqueous solution and representing the major focus of this investigation. These ions conform to the encounter complex of cis-[PtCl(NH3)2 (H2O)](+) with the incoming ligand and represent the first direct evidence of a prototypical Eigen-Wilkins encounter complex in solution, lying on the reaction coordinate for ligand substitution and extracted by ESI for mass spectrometric analysis. Activated [PtCl(NH3)2(L)(H2O)](+) ions dissociate by the loss of either H2O or L, the former process implying a ligand substitution event. IRMPD spectroscopy has thus revealed both structural details and reaction dynamics at the level of the isolated encounter complex.
The primary complex obtained from cisplatin and l-histidine in water has been detected and isolated by electrospray ionization. The so-obtained cis-[PtCl(NH ) (histidine)] complex has been characterized in detail by high-resolution mass spectrometry (MS), tandem MS, IR multiple photon dissociation (IRMPD) spectroscopy, and by quantum chemical calculations. The structural features revealed by IRMPD spectroscopy indicate that platinum binds to the imidazole group, which presents tautomeric forms. Thus, depending on the position of the amino acid pendant on the imidazole ring, isomeric complexes are formed that are remarkably different with respect to the ease with which they undergo fragmentation when activated either by energetic collisions or by multiple IR photon absorption. It is shown here how IRMPD kinetics can allow their relative proportions to be estimated.
Cisplatin and transplatin (cis- and trans-[PtCl(NH)]) have been allowed to react with methionine (Met) in water solution in a study aimed to characterize the monofunctional complex primarily formed. The thioether function of methionine is known to have a very high affinity for square planar platinum(ii) and sulfur-containing biomolecules have been proposed as a cisplatin drug reservoir on the way to platination at DNA. Both cisplatin and transplatin yield [PtCl(NH)Met] complexes, delivered by electrospray ionization in the gas phase and sampled as isolated species using tools based on mass spectrometry. The collision induced dissociation spectra of both cis-[PtCl(NH)Met] and trans-[PtCl(NH)Met] are quite similar and also the transport properties assayed by ion mobility mass spectrometry do not allow any appreciable discrimination. However, the vibrational spectra obtained by IR multiple photon absorption (IRMPD) spectroscopy show distinct features. Their analysis, supported by quantum chemical calculations, has revealed that while cisplatin attack is mainly directed to the sulfur atom of Met, transplatin shows a more balanced partition between sulfur and nitrogen binding. Among the vibrational signatures characterizing cis-[PtCl(NH)Met] and trans-[PtCl(NH)Met] complexes, the asymmetric NH stretching of the α-amino group of the amino acid at ca. 3440 cm is peculiar and diagnostic of S-platination. IRMPD kinetics evaluated at this frequency support the prevailing S-attack by cisplatin while approximately a 1 : 2 ratio of S- versus N-coordination is observed by transplatin, to be possibly related to the trans effect at the platinum center.
The binding motifs in the halide adducts with tyrosine ([Tyr + X], X = Cl, Br, I) have been investigated and compared with the analogues with 3-nitrotyrosine (nitroTyr), a biomarker of protein nitration, in a solvent-free environment by mass-selected infrared multiple photon dissociation (IRMPD) spectroscopy over two IR frequency ranges, namely 950-1950 and 2800-3700 cm. Extensive quantum chemical calculations at B3LYP, B3LYP-D3 and MP2 levels of theory have been performed using the 6-311++G(d,p) basis set to determine the geometry, relative energy and vibrational properties of likely isomers and interpret the measured spectra. A diagnostic carbonyl stretching band at ∼1720 cm from the intact carboxylic group characterizes the IRMPD spectra of both [Tyr + X] and [nitroTyr + X], revealing that the canonical isomers (maintaining intact amino and carboxylic functions) are the prevalent structures. The spectroscopic evidence reveals the presence of multiple non-covalent forms. The halide complexes of tyrosine conform to a mixture of plane and phenol isomers. The contribution of phenol-bound isomers is sensitive to anion size, increasing from chloride to iodide, consistent with the decreasing basicity of the halide, with relative amounts depending on the relative energies of the respective structures. The stability of the most favorable phenol isomer with respect to the reference plane geometry is in fact 1.3, -2.1, -6.8 kJ mol, for X = Cl, Br, I, respectively. The change in π-acidity by ring nitration also stabilizes anion-π interactions yielding ring isomers for [nitroTyr + X], where the anion is placed above the face of the aromatic ring.
The chemical properties accounting for the operation of a valuable matrix used in matrix assisted laser desorption ionization (MALDI) to perform mass spectrometry imaging (MSI), namely 3-(4,5bis(dimethylamino)napthalen-1-yl)furan-2,5-dione (4-maleicanhydridoproton sponge, MAPS), have been elucidated also by comparison with the parent molecule 1,8-Bis(dimethylamino)naphthalene (so-called Proton Sponge, PS). Both compounds present the bis(dimethylamino) groups, apt to efficiently trap a proton imparting positive charge. Only MAPS, though, owns the maleicanhydrido function acting as electrophile and yielding covalently bound adducts with a variety of analytes. In this way MAPS performs as 'carrier' for the analyte (A) of interest, at the same time minimizing the presence of useless, background ions. The covalent character of the adducts, [MAPS+H+A] + , is testified by their collision induced dissociation pattern, quite distinct from the one displayed by [PS+H] + , while PS does not form any [PS+H+A] + , thus confirming the key role of the maleicanhydrido functionality of MAPS. Vibrational spectroscopy of [MAPS+H+A] + adducts (A = H 2 O, NH 3) provided further structural evidence. The presence of a mobile proton on A was found to be a requisite for adduct formation by electrospray ionization of acetonitrile solutions, pointing to a possible role of MAPS in discriminating competing analytes based on molecular features. The performance of MAPS has been verified in MALDI-MSI of Atropa belladonna berries, exploiting MAPS binding to atropine.
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