Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 46
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Rapid Commun Mass Spectrom ; 33 Suppl 1: 28-34, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-29885203

RESUMO

RATIONALE: Among the sources of structural diversity in biomolecular ions, the co-existence of protomers is particularly difficult to take into account, which in turn complicates structural interpretation of gas-phase data. METHODS: We investigated the sensitivity of gas-phase photo-fragmentation measurements and ion mobility spectrometry (IMS) to the protonation state of a model peptide derivatized with chromophores. Accessible interconversion pathways between the different identified conformers were probed by tandem ion mobility measurement. Furthermore, the excitation coupling between the chromophores has been probed through photo-fragmentation measurements on mobility-selected ions. All results were interpreted based on molecular dynamics simulations. RESULTS: We show that protonation can significantly affect the photo-fragmentation yields. Especially, conformers with very close collision cross sections (CCSs) may display dramatically different photo-fragmentation yields in relation with different protonation patterns. CONCLUSIONS: We show that, even if precise structure assignment based on molecular modeling is in principle difficult for large biomolecular assemblies, the combination of photo-fragmentation and IMS can help to identify the signature of protomer co-existence for a population of biomolecular ions in the gas phase. Such spectroscopic data are particularly suitable to follow conformational changes.


Assuntos
Espectrometria de Mobilidade Iônica/métodos , Fotólise , Subunidades Proteicas , Simulação de Dinâmica Molecular , Peptídeos/análise , Peptídeos/química , Subunidades Proteicas/análise , Subunidades Proteicas/química , Espectrometria de Massas em Tandem/métodos
2.
J Phys Chem B ; 122(35): 8317-8329, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30068075

RESUMO

Unfolding of proteins gives detailed information about their structure and energetics and can be probed as a response to a change of experimental conditions. Ion mobility coupled to native mass spectrometry is a gas-phase technique that can observe such unfolding in the gas phase by monitoring the collision cross section (CCS) after applying an activation, for example, by collisions (collision-induced unfolding, CIU). The structural assignments needed to interpret the experiments can profit from dedicated modeling strategies. While predictions of ion-mobility data for well-defined and structurally characterized systems is straightforward, systematic free-energy calculations or biased molecular dynamics simulations that employ IMS data are still limited. The methods with which CCS values are calculated so far do not allow for analytical gradients needed in biased molecular dynamics (MD), and further, explicit CCS calculations still can pose computational bottleneck-when integrated into MD-bioinformatics workflows. These limitations motivate one to revisit known correlations of the CCS with the aim to find computationally cheap and versatile but still at least semiquantitative descriptions of the CCS by pure structural descriptors. We have therefore investigated the correlation of CCS with the key structural parameter often used in computational unfolding studies-the gyration radius-for several small monomeric and dimeric proteins. We work out the challenges and caveats of the combinations of the configurational sampling method and the CCS-calculation algorithm. The correlations were found to be sensitive to the generation conditions and additionally to the system topology. To reduce the amount of fitting to be undertaken, we devise a simple structural model for the CCS that shares some commonalities with the hard-sphere model and the projection algorithm but is designed to take unfolding into account. With this model, we suggest a two-point interpolating function rather than fitting a large data set, at only little deterioration of the predictive power. We further proceed to a model with composition and structure dependence that builds only upon the gyration radius and the chemical formula to apply the found CCS scaling behavior-the scaled macroscopic sphere (sMS) predictor. We demonstrate its applicability to describe unfolding and also its transferability for a larger set of structures from the RSCPDB. As we have found for the dimeric systems, that shape correlations with one global descriptor qualitatively break down, we finally suggest a recipe to switch between global and fragment-based CCS prediction, that takes up the ideas of coarse-graining protein complexes. The presented models and approaches might provide a basis to boost the integration of structural modeling with multistage IMS experiments, especially in the field of large-scale bioinformatics or "on-the-fly" biasing of MD, where computational efficiency is critical.

3.
J Am Soc Mass Spectrom ; 29(9): 1826-1834, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29949057

RESUMO

The nonapeptide oxytocin (OT) is used as a model sulfur-containing peptide to study the damage induced by vacuum UV (VUV) radiations. In particular, the effect of the presence (or absence in reduced OT) of oxytocin's internal disulfide bridge is evaluated in terms of photo-fragmentation yield and nature of the photo-fragments. Intact, as well as reduced, OT is studied as dianions and radical anions. Radical anions are prepared and photo-fragmented in two-color experiments (UV + VUV) in a linear ion trap. VUV photo-fragmentation patterns are analyzed and compared, and radical-induced mechanisms are proposed. The effect of VUV is principally to ionize but secondary fragmentation is also observed. This secondary fragmentation seems to be considerably enabled by the initial position of the radical on the molecule. In particular, the possibility to form a radical on free cysteines seems to increase the susceptibility to VUV fragmentation. Interestingly, disulfide bridges, which are fundamental for protein structure, could also be responsible for an increased resistance to ionizing radiations. Graphical Abstract.


Assuntos
Ânions/química , Dissulfetos/química , Ocitocina/química , Espectrometria de Massas , Oxirredução , Fotólise , Raios Ultravioleta , Vácuo
4.
Phys Chem Chem Phys ; 20(17): 12223-12228, 2018 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-29687123

RESUMO

Ruthenium complexes involving sulfoxide ligands can undergo linkage isomerization upon light absorption, accompanied by dramatic changes in their optical properties. These remarkable photochromic properties are sensitive to the nature of the ligand as well as to that of the solvent. We used tandem ion mobility spectrometry coupled to mass spectrometry to gain direct experimental insight into the isomerization pathways connecting the different linkage isomers of an isolated ruthenium complex with two dimethyl-sulfoxide ligands. We find that the isomerization behavior of the solvent-free complex differs from that previously reported in the solution-phase, which is in line with recent theoretical predictions.

5.
J Am Soc Mass Spectrom ; 29(2): 270-283, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-28980177

RESUMO

Mass spectrometry-based methods have made significant progress in characterizing post-translational modifications in peptides and proteins; however, certain aspects regarding fragmentation methods must still be improved. A good technique is expected to provide excellent sequence information, locate PTM sites, and retain the labile PTM groups. To address these issues, we investigate 10.6 µm IRMPD, 213 nm UVPD, and combined UV and IR photodissociation, known as HiLoPD (high-low photodissociation), for phospho-, sulfo-, and glyco-peptide cations. IRMPD shows excellent backbone fragmentation and produces equal numbers of N- and C-terminal ions. The results reveal that 213 nm UVPD and HiLoPD methods can provide diverse backbone fragmentation producing a/x, b/y, and c/z ions with excellent sequence coverage, locate PTM sites, and offer reasonable retention efficiency for phospho- and glyco-peptides. Excellent sequence coverage is achieved for sulfo-peptides and the position of the SO3 group can be pinpointed; however, widespread SO3 losses are detected irrespective of the methods used herein. Based on the overall performance achieved, we believe that 213 nm UVPD and HiLoPD can serve as alternative options to collision activation and electron transfer dissociations for phospho- and glyco-proteomics. Graphical Abstract ᅟ.


Assuntos
Glicopeptídeos/química , Peptídeos/química , Fosfopeptídeos/química , Processamento de Proteína Pós-Traducional , Enxofre/análise , Sequência de Aminoácidos , Raios Infravermelhos , Espectrometria de Massas/métodos , Fotólise , Raios Ultravioleta
6.
J Am Soc Mass Spectrom ; 29(1): 133-139, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29038996

RESUMO

In the context of native mass spectrometry, the development of gas-phase structural probes sensitive to the different levels of structuration of biomolecular assemblies is necessary to push forward conformational studies. In this paper, we provide the first example of the combination of ion mobility (IM) and Förster resonance energy transfer (FRET) measurements within the same experimental setup. The possibility to obtain mass- and mobility-resolved FRET measurements is demonstrated on a model peptide and applied to monitor the collision-induced unfolding of ubiquitin. Graphical Abstract ᅟ.

7.
Anal Chem ; 89(18): 9664-9670, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28727418

RESUMO

Using label-free ToF-SIMS imaging mass spectrometry, we generated a map of small molecules differentially expressed in the Drosophila wing imaginal disc. The distributions of these moieties were in line with gene expression patterns observed during wing imaginal disc development. Combining ToF-SIMS imaging and coherent anti-Stokes Raman spectroscopy (CARS) microspectroscopy allowed us to locally identify acylglycerols as the main constituents of the pattern differentiating the future body wall tissue from the wing blade tissue. The findings presented herein clearly demonstrate that lipid localization patterns are strongly correlated with a developmental gene expression. From this correlation, we hypothesize that lipids play a so far unrecognized role in organ development.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/genética , Perfilação da Expressão Gênica , Glicerídeos/análise , Discos Imaginais/crescimento & desenvolvimento , Espectrometria de Massa de Íon Secundário , Asas de Animais/crescimento & desenvolvimento , Animais , Drosophila melanogaster/anatomia & histologia , Glicerídeos/genética , Discos Imaginais/anatomia & histologia , Análise Espectral Raman , Fatores de Tempo , Asas de Animais/anatomia & histologia
8.
J Am Soc Mass Spectrom ; 28(10): 2181-2188, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28755260

RESUMO

The visible photodissociation mechanisms of QSY7-tagged peptides of increasing size have been investigated by coupling a mass spectrometer and an optical parametric oscillator laser beam. The experiments herein consist of energy resolved collision- and laser-induced dissociation measurements on the chromophore-tagged peptides. The results show that fragmentation occurs by similar channels in both activation methods, but that the branching ratios are vastly different. Observation of a size-dependent minimum laser pulse energy required to induce fragmentation, and collisional cooling rates in time resolved experiments show that laser-induced dissociation occurs through the absorption of multiple photons by the chromophore and the subsequent heating through vibrational energy redistribution. The differences in branching ratio between collision- and laser-induced dissociation can then be understood by the highly anisotropic energy distribution following absorption of a photon. Graphical Abstract ᅟ.


Assuntos
Espectrometria de Massas/métodos , Peptídeos/química , Íons/química , Sondas Moleculares/química , Fragmentos de Peptídeos/química , Processos Fotoquímicos , Fótons , Rodaminas/química
9.
Anal Chem ; 89(7): 4230-4237, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28263061

RESUMO

The shape of the spectral features in arrival time distributions (ATDs) recorded by ion mobility spectrometry (IMS) can often be interpreted in terms of the coexistence of different isomeric species. Interconversion between such species is also acknowledged to influence the shape of the ATD, even if no general quantitative description of this effect is available. We present an analytical model that allows simulating ATDs resulting from interconverting species. This model is used to reproduce experimental data obtained on a bistable system and to interpret discrepancies between measurements on different types of instruments. We show that the proposed model can be further exploited to extract kinetic and thermodynamic data from tandem-IMS measurements.

10.
Anal Chem ; 89(8): 4604-4610, 2017 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-28351129

RESUMO

To obtain a more detailed understanding of how structure influences the function and interaction of biomolecules, it is important to develop structure sensitive techniques to probe these relationships. Alongside in vivo and in vitro techniques, it is instructive to consider in vacuo methodologies: for example native mass spectrometry, ion mobility mass spectrometry, and FRET. Here, we propose a novel technique for probing biomolecular structure based on the changes in photophysics of a chromophore upon dimer formation. Comparison of solution and gas phase measurements on a doubly tagged tripeptide shows that dimer-induced fluorescence quenching is accompanied by an increase in photofragmentation yield. The 12-28 fragment of amyloid beta was used to show that as the charge state was increased-previously shown to cause a conformational change from compact random coil to extended helical structure-the disappearance of a band at 495 nm could be correlated with the level of self-quenching. The presence of features in the action spectrum of the +3 charge state of both quenched and unquenched chromophores allowed inference of multiple conformations. Single wavelength measurements on doubly tagged ubiquitin cations were performed to show that the technique is feasible on a small protein. These results demonstrate that self-quenching is a sensitive and fast gas-phase probe of biomolecular structure that can be directly linked to solution phase measurements. Further, it is capable of probing very small changes in conformation, making it complementary to FRET based techniques, which are insensitive at very short chromophore separations.


Assuntos
Peptídeos beta-Amiloides/química , Transferência Ressonante de Energia de Fluorescência , Peptídeos beta-Amiloides/metabolismo , Dimerização , Corantes Fluorescentes/química , Gases/química , Espectrometria de Mobilidade Iônica , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Estrutura Secundária de Proteína
11.
J Am Soc Mass Spectrom ; 28(1): 38-49, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27506208

RESUMO

Mass spectrometry is an extremely powerful technique for analysis of biological molecules, in particular proteins. One aspect that has been contentious is how much native solution-phase structure is preserved upon transposition to the gas phase by soft ionization methods such as electrospray ionization. To address this question-and thus further develop mass spectrometry as a tool for structural biology-structure-sensitive techniques must be developed to probe the gas-phase conformations of proteins. Here, we report Förster resonance energy transfer (FRET) measurements on a ubiquitin mutant using specific photofragmentation as a reporter of the FRET efficiency. The FRET data is interpreted in the context of circular dichroism, molecular dynamics simulation, and ion mobility data. Both the dependence of the FRET efficiency on the charge state-where a systematic decrease is observed-and on methanol concentration are considered. In the latter case, a decrease in FRET efficiency with methanol concentration is taken as evidence that the conformational ensemble of gaseous protein cations retains a memory of the solution phase conformational ensemble upon electrospray ionization. Graphical Abstract ᅟ.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Ubiquitina/química , Sequência de Aminoácidos , Animais , Cátions/química , Bovinos , Dicroísmo Circular , Gases/química , Simulação de Dinâmica Molecular , Mutação , Conformação Proteica , Espectrometria de Massas por Ionização por Electrospray , Eletricidade Estática , Ubiquitina/genética
12.
Chemphyschem ; 17(19): 3129-3138, 2016 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-27428813

RESUMO

Action spectroscopy has emerged as an analytical tool to probe excited states in the gas phase. Although comparison of gas-phase absorption properties with quantum-chemical calculations is, in principle, straightforward, popular methods often fail to describe many molecules of interest-such as xanthene analogues. We, therefore, face their nano- and picosecond laser-induced photofragmentation with excited-state computations by using the CC2 method and time-dependent density functional theory (TDDFT). Whereas the extracted absorption maxima agree with CC2 predictions, the TDDFT excitation energies are blueshifted. Lowering the amount of Hartree-Fock exchange in the DFT functional can reduce this shift but at the cost of changing the nature of the excited state. Additional bandwidth observed in the photofragmentation spectra is rationalized in terms of multiphoton processes. Observed fragmentation from higher-lying excited states conforms to intense excited-to-excited state transitions calculated with CC2. The CC2 method is thus suitable for the comparison with photofragmentation in xanthene analogues.

13.
J Am Soc Mass Spectrom ; 27(9): 1435-42, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27287047

RESUMO

Herein we report the successful implementation of the consecutive and simultaneous photodissociation with high (213 nm) and low (10.6 µm) energy photons (HiLoPD, high-low photodissociation) on ubiquitin in a quadrupole-Orbitrap mass spectrometer. Absorption of high-energy UV photon is dispersed over the whole protein and stimulates extensive C-Cα backbone fragmentation, whereas low-energy IR photon gradually increases the internal energy and thus preferentially dissociates the most labile amide (C-N) bonds. We noticed that simultaneous irradiation of UV and IR lasers on intact ubiquitin in a single MS/MS experiment provides a rich and well-balanced fragmentation array of a/x, b/y, and z ions. Moreover, secondary fragmentation from a/x and z ions leads to the formation of satellite side-chain ions (d, v, and w) and can help to distinguish isomeric residues in a protein. Implementation of high-low photodissociation in a high-resolution mass spectrometer may offer considerable benefits to promote a comprehensive portrait of protein characterization. Graphical Abstract ᅟ.

14.
J Phys Chem A ; 120(20): 3484-90, 2016 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-27118657

RESUMO

The use of the xanthene family of dyes as fluorescent probes in a wide range of applications has provided impetus for the studying of their photophysical properties. In particular, recent advances in gas-phase techniques such as FRET that utilize such chromophores have placed a greater importance on the characterization of these properties in the gas phase. Additionally, the use of synthetic linker chains to graft the chromophores in a site-specific manner to their target system is ubiquitous. There is, however, often limited information on how the addition of such a linker chain may affect the photophysical properties of the chromophores, which is of fundamental importance for interpretation of experimental data reliant on grafted chromophores. Here, we present data on the optical spectroscopy of different protonation states of Eosin Y, a fluorescein derivative. We compare the photophysics of Eosin Y to its maleimide conjugate, and to the thioether product of the reaction of this conjugate with cysteamine. Comparison of the mass spectra following laser irradiation shows that very different relaxation takes place upon addition of the maleimide moiety but that the photophysics of the bare chromophore are restored upon addition of cysteamine. This radical change in the photophysics is interpreted in terms of charge-transfer states, whose energy relative to the S1 ← S0 transition of the chromophore is dependent on the conjugation of the maleimide moiety. We also show that the shape of the absorption band is unchanged in the gas-phase as compared to the solution-phase, showing a maximum with a shoulder toward the blue, and examination of isotope distributions of the isolated ions show that this shoulder cannot be due to the presence of dimers. Consideration of the fluorescence emission spectrum allows a tentative assignment of the shoulder to be due to a vibrational progression with a high Franck-Condon factor.

15.
Phys Chem Chem Phys ; 18(13): 9061-9, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26972232

RESUMO

We present theoretical modelling, ion mobility spectrometry and action-FRET experiments for chromophore-grafted amyloid-ß(12-28) dimers. A first-principles global minimum search based on replica-exchange molecular dynamics (REMD) leads to a compact structure with strong interstrand interactions. We use REMD with a distance restraint that implements an adaptive effective bias upon average FRET-efficiencies and thus guides the sampling by the action-FRET measurement. This procedure leads to a pair of weakly interacting peptides. Ion-mobility confirms that the weakly interacting structure and not the global minimum with strongly interacting peptides is populated in the experiment. The presence of a high energy barrier between the two structural families, as evidenced from the MD data, suggests that a kinetically trapped structure is observed in the experiment.


Assuntos
Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/química , Transferência Ressonante de Energia de Fluorescência , Conformação Proteica
16.
J Am Chem Soc ; 138(13): 4401-7, 2016 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-26974184

RESUMO

Charge transfer mechanisms lay at the heart of chemistry and biochemistry. Proton coupled electron transfers (PCET) are central in biological processes such as photosynthesis and in the respiratory chain, where they mediate long-range charge transfers. These mechanisms are normally difficult to harness experimentally due to the intrinsic complexity of the associated biological systems. Metal-peptide cations experience both electron and proton transfers upon photoexcitation, proving an amenable model system to study PCET. We report on a time-resolved experiment designed to follow this dual charge transfer kinetics in [HG3W+Ag](+) (H = histidine, G = glycine, W = tryptophan) on time scales ranging from femtoseconds to milliseconds. While electron transfer completes in less than 4 ps, it triggers a proton transfer lasting over hundreds of microseconds. Molecular dynamics simulations show that conformational dynamic plays an important role in slowing down this reaction. This combined experimental and computational approach provides a view of PCET as a single phenomenon despite its very wide time-domain span.

17.
J Phys Chem B ; 120(4): 709-14, 2016 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-26756462

RESUMO

Monitoring the chromism induced by intramolecular hydrogen and charge transfers within proteins as well as the isomerization of both protein and cofactor is essential not only to understand photoactive signaling pathways but also to design targeted opto-switchable proteins. We used a dual-ion mobility drift tube coupled to a tunable picosecond laser to explore the optical and structural properties of a peptide chain bound to a chromophore-a prototype system allowing for a proton transfer coupled to conformational change. With the support of molecular dynamics and DFT calculations, we show how proton transfer between the peptide and its cofactor can dramatically modify the optical properties of the system and demonstrate that these changes can be triggered by collisional activation in the gas phase.


Assuntos
Cor , Peptídeos/química , Conformação Proteica , Isomerismo
19.
J Am Soc Mass Spectrom ; 27(3): 474-86, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26545767

RESUMO

Characterization of acidic peptides and proteins is greatly hindered due to lack of suitable analytical techniques. Here we present the implementation of 213 nm ultraviolet photodissociation (UVPD) in high-resolution quadrupole-Orbitrap mass spectrometer in negative polarity for peptide anions. Radical-driven backbone fragmentation provides 22 distinctive fragment ion types, achieving the complete sequence coverage for all reported peptides. Hydrogen-deficient radical anion not only promotes the cleavage of Cα-C bond but also stimulates the breaking of N-Cα and C-N bonds. Radical-directed loss of small molecules and specific side chain of amino acids are detected in these experiments. Radical containing side chain of amino acids (Tyr, Ser, Thr, and Asp) may possibly support the N-Cα backbone fragmentation. Proline comprising peptides exhibit the unusual fragment ions similar to reported earlier. Interestingly, basic amino acids such as Arg and Lys also stimulated the formation of abundant b and y ions of the related peptide anions. Loss of hydrogen atom from the charge-reduced radical anion and fragment ions are rationalized by time-dependent density functional theory (TDDFT) calculation, locating the potential energy surface (PES) of ππ* and repulsive πσ* excited states of a model amide system.


Assuntos
Peptídeos/química , Fotólise , Sequência de Aminoácidos , Íons/química , Espectrometria de Massas , Modelos Moleculares , Raios Ultravioleta
20.
Phys Chem Chem Phys ; 18(3): 1807-17, 2016 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-26679547

RESUMO

Ion mobility experiments are combined with Infra-Red Multiple Photon Dissociation (IRMPD) spectroscopy and quantum chemical calculations for assessing the role of chirality in the structure of protonated and sodiated di- or tetra-peptides. Sodiated systems show a strong chirality dependence of the competition between Na(+)O and Na(+)π interactions. Chirality effects are more subtle in protonated systems and manifest themselves by differences in the secondary interactions such hydrogen bonds between neutral groups or those involving the aromatic rings.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA