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1.
Chembiochem ; 24(24): e202300688, 2023 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-37815502

RESUMO

Target-directed dynamic combinatorial chemistry is a very attractive strategy for the discovery of bioactive peptides. However, its application has not yet been demonstrated, presumably due to analytical challenges that arise from the diversity of a peptide library with combinatorial side-chains. We previously reported an efficient method to generate, under biocompatible conditions, large dynamic libraries of cyclic peptides grafted with amino acid's side-chains, by thiol-to-thioester exchanges. In this work, we present analytical tools to easily characterize such libraries by HPLC and mass spectrometry, and in particular to simplify the isomers' distinction requiring sequencing by MS/MS fragmentations. After structural optimization, the cyclic scaffold exhibits a UV-tag, absorbing at 415 nm, and an ornithine residue which favors the regioselective ring-opening and simultaneous MS/MS fragmentation, in the gas-phase.


Assuntos
Técnicas de Química Combinatória , Peptídeos Cíclicos , Peptídeos Cíclicos/química , Espectrometria de Massas em Tandem , Biblioteca de Peptídeos , Peptídeos
2.
Rapid Commun Mass Spectrom ; 37(8): e9476, 2023 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-36656736

RESUMO

RATIONALE: Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) is an approach derived from matrix-assisted laser desorption/ionization (MALDI)-MS which overcomes the drawbacks associated with the use of organic matrices required to co-crystallize with the analytes. Indeed, nanomaterials commonly used in SALDI-MS as inert surfaces to promote desorption/ionization (D/I) ensure straightforward direct deposition of samples while providing mass spectra with ions only related to the compound of interest. The objective of this study was to develop a novel SALDI-MS approach based on steel plates that are surfaces very rapidly and easily tuned to perform the most efficient peptide detection as possible. To compare the SALDI efficacy of such metal substrates, D/I efficiency and deposit homogeneity were evaluated according to steel plate fabrication processes. METHODS: The studied surfaces were nanostructured steel plates that were chemically modified by perfluorosilane and textured according to different frequencies and laser writing powers. The capacity of each tested 100 surfaces was demonstrated by comparative analyses of a mixture of standard peptides (m/z 600-3000) performed with a MALDI-TOF instrument enabling MALDI, SALDI and imaging experiments. RESULTS: A peptide mix was used to screen the different surfaces depending on their D/I efficiency and their ability to ensure homogeneous deposit of the samples. For that purpose, deposition homogeneity was visualized owing to reconstructed ionic images from all protonated or sodiated ions of the 10 peptides constituting the standard mix. CONCLUSIONS: Seven surfaces were then selected satisfying the required D/I efficiency and deposit homogeneity criteria. Results obtained with these optimal surfaces were then compared with those recorded by MALDI-MS analyses used as references.


Assuntos
Nanoestruturas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Nanoestruturas/química , Peptídeos , Lasers , Íons
3.
Toxins (Basel) ; 14(11)2022 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-36422973

RESUMO

Animal venoms are a rich source of pharmacological compounds with ecological and evolutionary significance, as well as with therapeutic and biotechnological potentials. Among the most promising venomous animals, cone snails produce potent neurotoxic venom to facilitate prey capture and defend against aggressors. Conus striatus, one of the largest piscivorous species, is widely distributed, from east African coasts to remote Polynesian Islands. In this study, we investigated potential intraspecific differences in venom composition between distinct geographical populations from Mayotte Island (Indian Ocean) and Australia (Pacific Ocean). Significant variations were noted among the most abundant components, namely the κA-conotoxins, which contain three disulfide bridges and complex glycosylations. The amino acid sequence of a novel κA-conotoxin SIVC, including its N-terminal acetylated variant, was deciphered using tandem mass spectrometry (MS/MS). In addition, the glycosylation pattern was found to be consisting of two HexNAc and four Hex for the Mayotte population, which diverge from the previously characterized two HexNAc and three Hex combinations for this species, collected elsewhere. Whereas the biological and ecological roles of these modifications remain to be investigated, population-specific glycosylation patterns provide, for the first time, a new level of intraspecific variations in cone snail venoms.


Assuntos
Conotoxinas , Caramujo Conus , Animais , Caramujo Conus/química , Conotoxinas/química , Peçonhas/metabolismo , Proteômica , Espectrometria de Massas em Tandem
4.
Traffic ; 23(5): 287-304, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35466519

RESUMO

Proteasomes are major non-lysosomal proteolytic complexes localized in the cytoplasm and in the nucleus of eukaryotic cells. Strikingly, high levels of extracellular proteasome have also been evidenced in the plasma (p-proteasome) of patients with specific diseases. Here, we examined the process by which proteasomes are secreted, as well as their structural and functional features once in the extracellular space. We demonstrate that assembled 20S core particles are secreted by cells within microvesicles budding from the plasma membrane. Part of the extracellular proteasome pool is also free of membranes in the supernatant of cultured cells, and likely originates from microvesicles leakage. We further demonstrate that this free proteasome released by cells (cc-proteasome for cell culture proteasome) possesses latent proteolytic activity and can degrade various extracellular proteins. Both standard (no immune-subunits) and intermediate (containing some immune-subunits) forms of 20S are observed. Moreover, we show that galectin-3, which displays a highly disordered N-terminal region, is efficiently cleaved by purified cc-proteasome, without SDS activation, likely after its binding to PSMA3 (α7) subunit through its intrinsically disordered region. As a consequence, galectin-3 is unable to induce red blood cells agglutination when preincubated with cc-proteasome. These results highlight potential novel physio- and pathologic functions for the extracellular proteasome.


Assuntos
Galectina 3 , Complexo de Endopeptidases do Proteassoma , Aglutinação , Citoplasma/metabolismo , Galectina 3/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise
5.
Chemistry ; 28(36): e202200454, 2022 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-35394670

RESUMO

An efficient strategy for the synthesis of large libraries of conformationally defined peptides is reported, using dynamic combinatorial chemistry as a tool to graft amino acid side chains on a well-ordered 3D (3-dimension) peptide backbone. Combining rationally designed scaffolds with combinatorial side chains selection represents an alternative method to access peptide libraries for structures that are not genetically encodable. This method would allow a breakthrough for the discovery of protein mimetic for unconventional targets for which little is known.


Assuntos
Aminoácidos , Biblioteca de Peptídeos , Técnicas de Química Combinatória/métodos , Peptídeos/química , Proteínas
6.
J Chromatogr A ; 1648: 462213, 2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-33991752

RESUMO

Comprehensive characterization of physicochemical properties of monoclonal antibodies (mAbs) is a critical process to ensure their quality, efficacy, and safety. For this purpose, mAb analysis at different levels (bottom-up, middle-up) is a common approach that includes rather complex multistep sample preparation (reduction, digestion). To ensure high analysis performance, the development of fully integrated methodologies is highly valuable. Capillary zone electrophoresis is a particularly well-adapted technique for the multistep implementation of analytical strategies from sample preparation to detection. This feature was employed to develop novel integrated methodologies for the analysis of mAb at the middle-up level. Multiple in-line reactions (simultaneous reduction and digestion) were performed for the first time in the separation capillary. Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) was used as an effective reducing agent under a broad pH range and IdeS (Immunoglobulin degrading enzyme from Streptococcus) as a highly specific enzyme for mAb digestion. Transverse diffusion of laminar flow profile (TDLFP) was applied for reactants mixing. Both in-line sample preparation and separation parameters were optimized under non-denaturing and denaturing conditions. The developed in-line methodologies provided good reproducibility and higher peak efficiencies comparing with off-line assays. They were successfully applied to different mAbs.


Assuntos
Anticorpos Monoclonais/análise , Eletroforese Capilar/métodos , Difusão , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
7.
Electrophoresis ; 42(11): 1229-1237, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33650106

RESUMO

For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom-up level to generate specific fingerprints that can be used to tackle post-translational modifications or ensure production consistency between lots. To circumvent time-consuming and labor-intensive off-line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in-line methodology for the routine quality control of mAbs at the bottom-up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in-line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In-line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS-compatible BGE was used to obtain high resolution separation of released peptides and in-line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE-fingerprints were successfully established for different mAbs and an antibody-drug conjugate.


Assuntos
Anticorpos Monoclonais , Anticorpos Monoclonais/análise , Eletroforese Capilar , Tensoativos , Tripsina
8.
J Pharm Biomed Anal ; 188: 113438, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32623316

RESUMO

The CDK4/6 inhibitors palbociclib and ribociclib are kinase inhibitors used in association with hormonal therapy for the management of patients with metastatic breast cancer. Like most kinase inhibitors, therapeutic drug monitoring may be used for personalize their dosage. To this aim, we developed and validated a sensitive and specific HPLC-MS/MS method for palbociclib and ribociclib quantification in blood samples. We then quantified exposure to palbociclib (plasma trough concentration; Ctrough) in a real-life cohort of patients with locally invasive or metastatic breast cancer (n = 18) at day 15 of the first cycle of palbociclib treatment to characterize palbociclib concentration at steady state (Clinicaltrials.gov identifier NCT04025541, IdRCB n° 2018-A00064-51, 03/07/2018). The geometric mean (± standard deviation [min-max]) of palbociclib plasma Ctrough was 88.58 ng/mL (± 26.4 [46.5 ng/mL - 133 ng/mL]) at day 15. Some covariates, such as drug-drug interactions, could explain the concentration variations observed in our Caucasian cohort. These first results in real-life settings obtained with our HPLC-MS/MS method give important information on palbociclib monitoring and pharmacokinetic variability.


Assuntos
Neoplasias da Mama , Preparações Farmacêuticas , Benzimidazóis , Neoplasias da Mama/tratamento farmacológico , Cromatografia Líquida , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Interações Medicamentosas , Feminino , Humanos , Inibidores de Proteínas Quinases , Espectrometria de Massas em Tandem
9.
Analyst ; 145(5): 1759-1767, 2020 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-31913378

RESUMO

Monoclonal antibodies (mAbs) are undergoing rapid growth in the pharmaceutical industry due to their clinical efficiency. Concomitantly, robust, cost-effective, and high throughput analytical methods are needed for their quality control. Among all analytical techniques, capillary electrophoresis (CE) presents alternative and attractive features because the capillary can be used both as a microreactor and as a support for separation. Transverse diffusion of laminar flow profiles was applied for the middle-up analysis of mAbs for the first time. Infliximab was selected as the model mAb. All middle-up analysis steps (enzymatic digestion, electrophoretic separation and UV detection) were integrated into the same capillary. The conditions for the separation of infliximab subunits (pH, ionic strength, and type of background electrolyte) and in-line digestion parameters (reactant injection conditions, time, temperature and enzyme/mAb ratio) were optimized. The in-line methodology was compared to the off-line methodology and evaluated in terms of proteolysis efficiency, repeatability, and applicability to different mAbs. Finally, the methodology was transferred to capillary electrophoresis coupled to mass spectrometry (sheathless interface) to identify infliximab subunits. The in-line methodology was successfully implemented with a simplified injection scheme, temperature control, fast enzymatic reaction and high resolution of separation of infliximab subunits under pseudo-native MS compatible conditions. In comparison with the off-line methodology, reactant consumption was reduced by a factor of 1000, and the numbers of theoretical plates were increased by a factor of 2.


Assuntos
Anticorpos Monoclonais Humanizados/análise , Eletroforese Capilar/métodos , Subunidades Proteicas/análise , Ribonuclease Pancreático/química , Animais , Anticorpos Monoclonais Humanizados/química , Bovinos , Eletroforese Capilar/instrumentação , Subunidades Proteicas/química , Proteólise
10.
J Mass Spectrom ; 51(6): 412-23, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27270864

RESUMO

The preparation of tropical wood surface sections for time-of-flight secondary ion mass spectrometry imaging is described, and the use of delayed extraction of secondary ions and its interest for the analysis of vegetal surface are shown. The method has been applied to the study by time-of-flight secondary ion mass spectrometry imaging with a resolution of less than one micron of a tropical wood species, Dicorynia guianensis, which is one of the most exploited wood in French Guiana for its durable heartwood. The heartwood of this species exhibits an economical importance, but its production is not controlled in forestry. Results show an increase of tryptamine from the transition zone and a concomitant decrease of inorganic ions and starch fragment ions. These experiments lead to a better understanding of the heartwood formation and the origin of the natural durability of D. guianensis. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Fabaceae/química , Espectrometria de Massa de Íon Secundário/métodos , Madeira/química , Fabaceae/ultraestrutura , Imagem Óptica/métodos , Dióxido de Silício/análise , Amido/análise , Triptaminas/análise , Madeira/ultraestrutura
11.
FASEB J ; 28(6): 2603-19, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24604079

RESUMO

A new approach to treating Duchenne muscular dystrophy was investigated by using the ester or amide covalent association of arginine [nitric oxide (NO) pathway] and butyrate [histone deacetylase (HDAC) inhibition] in mdx mice and patient myotubes. Two prodrugs were synthesized, and the beneficial effects on dystrophic phenotype were studied. Nerve excitability abnormalities detected in saline-treated mice were almost totally rescued in animals treated at low doses (50-100 mg/kg/d). Force and fatigue resistance were improved ≈60% and 3.5-fold, respectively, and the percentage of necrosis in heart sections was reduced ≈90% in the treated mice. A decrease of >50% in serum creatine kinase indicated an overall improvement in the muscles. Restoration of membrane integrity was studied directly by measuring the reduction (≈74%) of Evans blue incorporation in the limb muscles of the treated animals, the increase in utrophin level, and the normalization of lipid composition of the heart. In cultures of human myotubes (primary cells and cell line), both prodrugs and HDAC inhibitors increased by 2- to 4-fold the utrophin level, which was correctly localized at the membrane. ß-Dystroglycan and embryonic myosin protein levels were also increased. Finally, a 50% reduction in the number of spontaneous Ca(2+) spikes was observed after treatment with NO synthase substrate and HDAC inhibitors. Overall, the beneficial effects were obtained with doses 10 (in vivo) and 5 (in vitro) times lower than those of the salt formulation. Altogether, these data constitute proof of principle of the beneficial effects of low doses of arginine butyrate derivatives on muscular dystrophy, enhancing the NO pathway and inhibiting HDAC.


Assuntos
Arginina/análogos & derivados , Butiratos/uso terapêutico , Distrofia Muscular Animal/tratamento farmacológico , Distrofia Muscular de Duchenne/tratamento farmacológico , Animais , Arginina/uso terapêutico , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/farmacologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos mdx , Fibras Musculares Esqueléticas/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculos/efeitos dos fármacos , Músculos/fisiologia , Utrofina/metabolismo
12.
Int J Biochem Cell Biol ; 52: 77-82, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24513532

RESUMO

A consistent body of research has linked cystic fibrosis (CF) with variations in the tissue and fluid content in a number of lipid molecules. However, little is known about the spatial localization of those variations. We have recently applied TOF-SIMS mass spectrometry imaging to detect differential lipid signatures at the colon epithelium between normal and cftr-/- mice. In the present work we have used this technology to investigate potential differences in the spatial distribution of lipids due to Pseudomonas aeruginosa (P.a.) infection in mouse lung expressing or not cftr. Wild-type and exon 10 cftr knockout mice were subjected to intranasal infection with a clinical strain of P.a. Lung cryosections from infected and non-infected mice were subjected to cluster TOF-SIMS analysis in the negative ion mode. We observed a highly specific localization of a phosphoinositol fragment ion at m/z 299.1 in bronchial epithelium. Using this ion to delineate a region of interest, we studied the relative abundance of ions below m/z 1500. We found a significant increase in m/z 465.4 (identified as cholesteryl sulfate) in cftr-/- epithelium and in response to bacterial infection, as well as a decrease in most carboxylic ions. In conclusion, the m/z 299.1 ion can be used as a marker of bronchial epithelium, where P.a. infection leads to increased presence of cholesteryl sulfate in this tissue. TOF-SIMS imaging reveals as a valuable tool for the study of respiratory epithelium.


Assuntos
Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Animais , Ésteres do Colesterol/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Modelos Animais de Doenças , Metabolismo dos Lipídeos , Pulmão/metabolismo , Pulmão/microbiologia , Espectrometria de Massas/métodos , Metabolômica/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Pseudomonas/microbiologia
13.
Biointerphases ; 10(1): 018901, 2014 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25708628

RESUMO

Imaging by secondary ion mass spectrometry coupled to time-of-flight mass analysis (TOF-SIMS) is a method of which the applications have greatly increased since 10 years. Taking advantage of the development of cluster ion sources, TOF-SIMS offers images of molecular ions at a micrometer lateral resolution or slightly below and does not require complex sample preparation. Although TOF-SIMS has been primarily dedicated to surface analysis of inorganic or polymeric samples, several groups have successfully demonstrated that TOF-SIMS imaging is also perfectly suited for mapping organic compounds, such as drugs or lipids, directly on tissue sections from animals or from human biopsies. This minireview will enlighten some of these developments in the field of biomedical applications.


Assuntos
Pesquisa Biomédica/métodos , Patologia/métodos , Espectrometria de Massa de Íon Secundário/métodos , Animais , Pesquisa Biomédica/tendências , Humanos , Patologia/tendências
14.
Mass Spectrom Rev ; 33(6): 442-51, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24265115

RESUMO

Recent developments in instrumentation, ion beams or analyzers, for cluster time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging are described here. The methods which are employed to increase the sensitivity or to perform three-dimensional analyses in the organic materials are also illustrated. This review shows the improvements made for lipid imaging by cluster TOF-SIMS in various types of material and applications, and gives reasons for the expansion of its utilization.


Assuntos
Aumento da Imagem/instrumentação , Metabolismo dos Lipídeos/fisiologia , Imageamento por Ressonância Magnética/instrumentação , Espectroscopia de Ressonância Magnética/instrumentação , Microquímica/instrumentação , Imagem Molecular/instrumentação , Animais , Desenho de Equipamento , Humanos , Distribuição Tecidual
15.
Anal Chem ; 85(16): 7745-52, 2013 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-23875833

RESUMO

Argon cluster ion sources for sputtering and secondary ion mass spectrometry use projectiles consisting of several hundreds of atoms, accelerated to 10-20 keV, and deposit their kinetic energy within the top few nanometers of the surface. For organic materials, the sputtering yield is high removing material to similar depth. Consequently, the exposed new surface is relatively damage free. It has thus been demonstrated on model samples that it is now really possible to perform dual beam depth profiling experiments in organic materials with this new kind of ion source. Here, this possibility has been tested directly on tissue samples, 14 µm thick rat brain sections, allowing primary ion doses much larger than the so-called static secondary ion mass spectrometry (SIMS) limit and demonstrating the possibility to enhance the sensitivity of time-of-flight (TOF)-SIMS biological imaging. However, the depth analyses have also shown some variations of the chemical composition as a function of depth, particularly for cholesterol, as well as some possible matrix effects due to the presence or absence of this compound.


Assuntos
Argônio/química , Encéfalo/metabolismo , Lipídeos/normas , Animais , Íons , Ratos , Espectrometria de Massa de Íon Secundário
16.
Acta Neuropathol ; 125(1): 133-44, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22956244

RESUMO

Although cholesterol has been involved in the pathophysiology of Alzheimer disease (AD), its distribution in the cerebral cortex over the course of AD is unknown. We describe an original method to quantify cholesterol distribution using time-of-flight secondary ion mass spectrometry imaging. Cholesterol was unevenly distributed along the cortical thickness, being more abundant close to the white matter, in both control and AD cases. However, the mean cholesterol signal was significantly higher in the lower half of the cortex in AD samples compared to controls. This increase, when converted into cortical layers, was statistically significant for layers III and IV and did not reach significance in layers V + VI, the variability being too high at the interface between grey and white matter. The density of neurofibrillary tangles and of senile plaques was not statistically linked to the abundance of cholesterol. Cholesterol overload thus appears a new and independent alteration of AD cerebral cortex. The structure in which cholesterol accumulates and the mechanism of this accumulation remain to be elucidated.


Assuntos
Doença de Alzheimer/metabolismo , Córtex Cerebral/metabolismo , Colesterol/metabolismo , Emaranhados Neurofibrilares/patologia , Espectrometria de Massa de Íon Secundário , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/patologia , Córtex Cerebral/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Emaranhados Neurofibrilares/metabolismo , Neuroimagem , Placa Amiloide , Espectrometria de Massa de Íon Secundário/métodos
17.
J Am Soc Mass Spectrom ; 21(4): 635-45, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20097575

RESUMO

Nuclear receptors, such as the retinoic acid receptor (RAR) or the 9-cis retinoic acid receptor (RXR), interact not only with their ligands but also with other types of receptors and with DNA. Here, two complementary mass spectrometry (MS) methods were used to study the interactions between retinoic receptors (RXR/RAR) and DNA: non-denaturing nano-electrospray (nanoESI MS), and high-mass matrix-assisted laser desorption ionization (MALDI MS) combined with chemical cross-linking. The RAR x RXR heterodimer was studied in the presence of a specific DNA sequence (DR5), and a specific RAR x RXR x DNA complex was detected with both MS techniques. RAR by itself showed no significant homodimerization. A complex between RAR and the double stranded DR5 was detected with nanoESI. After cross-linking, high-mass MALDI mass spectra showed that the RAR binds the single stranded DR5, and the RAR dimer binds both single and double stranded DR5. Moreover, the MALDI mass spectrum shows a larger RAR dimer signal in the presence of DNA. These results suggest that a gene-regulatory site on DNA can induce quaternary structural changes in a transcription factor such as RAR.


Assuntos
DNA/química , Nanotecnologia/métodos , Receptores do Ácido Retinoico/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sítios de Ligação , DNA/análise , Ligação Proteica , Receptores do Ácido Retinoico/análise
18.
J Am Soc Mass Spectrom ; 21(2): 286-9, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19931466

RESUMO

The study of noncovalent interactions by mass spectrometry has become an active field of research in recent years. The role of the different noncovalent intermolecular forces is not yet fully understood since they tend to be modulated upon transfer into the gas phase. The hydrophobic effect, which plays a major role in protein folding, adhesion of lipid bilayers, etc., is absent in the gas phase. Here, noncovalent complexes with different types of interaction forces were investigated by mass spectrometry and compared with the complex present in solution. Creatine kinase (CK), glutathione S-transferase (GST), ribonuclease S (RNase S), and leucine zipper (LZ), which have dissociation constants in the nM range, were studied by native nanoelectrospray mass spectrometry (nanoESI-MS) and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking (XL). Complexes interacting with hydrogen bonds survived the transfer into gas phase intact and were observed by nanoESI-MS. Complexes that are bound largely by the hydrophobic effect in solution were not detected or only at very low intensity. Complexes with mixed polar and hydrophobic interactions were detected by nanoESI-MS, most likely due to the contribution from polar interactions. All noncovalent complexes could easily be studied by XL MALDI-MS, which demonstrates that the noncovalently bound complexes are conserved, and a real "snap-shot" of the situation in solution can be obtained.


Assuntos
Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares
19.
Anal Chem ; 82(1): 172-9, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19994840

RESUMO

Chemical cross-linking of proteins permits the stabilization of noncovalent complexes through introduction of covalent bonds. A crucial challenge is to find the fastest and most efficient cross-linkers in order to minimize reaction times and to handle delicate complexes. New cross-linkers were synthesized by introducing N-hydroxyphthalimide, hydroxybenzotriazole, and 1-hydroxy-7-azabenzotriazole as leaving groups instead of the commonly used N-hydroxysuccimidyl moiety. With the use of matrix-assisted laser desorption ionization (MALDI) mass spectrometry, these new cross-linkers were then compared with the commercially available disuccinimidyl suberate (DSS) for covalent stabilization of the gluthatione-S-transferase (GST) dimer and of an antibody-antigen complex. They showed a better efficiency, generated about 30% more cross-linked complex, and reacted about 10 times faster than DSS. The reaction with the GST dimer was utilized to get information about their reaction efficiency and kinetics. Their ability to stabilize only specific protein complexes was verified by incubating them with a mixture of the proteins GST and ubiquitin. Finally, the cross-linkers were incubated with synthetic peptides to study the selectivity of the binding with various amino acid side chains. Not only lysine but also tyrosine was found to react with the newly synthesized cross-linker containing 1-hydroxy-7-azabenzotriazole as the reactive group.


Assuntos
Aminas/química , Anticorpos/química , Glutationa Transferase/química , Príons/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos
20.
Curr Opin Struct Biol ; 19(5): 632-9, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19782560

RESUMO

Mass spectrometry is becoming a more and more powerful tool for investigating protein complexes. Recent developments, based on different ionization techniques, electrospray, desorption/ionization and others are contributing to the usefulness of MS to describe the organization and structure of large non-covalent assemblies.


Assuntos
Espectrometria de Massas/métodos , Multimerização Proteica , Proteínas/análise , Animais , Humanos , Cinética , Proteínas/química , Proteínas/metabolismo , Termodinâmica
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