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1.
Clin Proteomics ; 20(1): 26, 2023 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-37393264

RESUMO

We have developed a rapid and highly specific assay for detecting and monitoring SARS-CoV-2 infections by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). As MALDI-TOF mass spectrometers are available in a clinical setting, our assay has the potential to serve as alternative to the commonly used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Sample preparation prior to MALDI-TOF-MS involves the tryptic digestion of SARS-CoV-2 proteins, followed by an enrichment of virus-specific peptides from SARS-CoV-2 nucleoprotein via magnetic antibody beads. Our MALDI-TOF-MS method allows the detection of SARS-CoV-2 nucleoprotein in sample collection medium as low as 8 amol/µl. MALDI-TOF mass spectra are obtained in just a few seconds, which makes our MS-based assay suitable for a high-throughput screening of SARS-CoV-2 in healthcare facilities in addition to PCR. Due to the specific detection of virus peptides, different SARS-CoV-2 variants are readily distinguished from each other. Specifically, we show that our MALDI-TOF-MS assay discriminates SARS-CoV-2 strain B.1.617.2 "delta variant" from all other variants in patients' samples, making our method highly valuable to monitor the emergence of new virus variants.

2.
Sci Rep ; 13(1): 8497, 2023 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-37231156

RESUMO

The tetrameric tumor suppressor p53 represents a great challenge for 3D-structural analysis due to its high degree of intrinsic disorder (ca. 40%). We aim to shed light on the structural and functional roles of p53's C-terminal region in full-length, wild-type human p53 tetramer and their importance for DNA binding. For this, we employed complementary techniques of structural mass spectrometry (MS) in an integrated approach with computational modeling. Our results show no major conformational differences in p53 between DNA-bound and DNA-free states, but reveal a substantial compaction of p53's C-terminal region. This supports the proposed mechanism of unspecific DNA binding to the C-terminal region of p53 prior to transcription initiation by specific DNA binding to the core domain of p53. The synergies between complementary structural MS techniques and computational modeling as pursued in our integrative approach is envisioned to serve as general strategy for studying intrinsically disordered proteins (IDPs) and intrinsically disordered region (IDRs).


Assuntos
Proteínas Intrinsicamente Desordenadas , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/metabolismo , Simulação por Computador , Proteínas Intrinsicamente Desordenadas/química , DNA/metabolismo , Espectrometria de Massas , Ligação Proteica
3.
PLoS One ; 18(3): e0282593, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36867608

RESUMO

We show the effects of the three purine derivatives, caffeine, theophylline, and istradefylline, on cAMP production by adenylyl cyclase 5 (ADCY5)-overexpressing cell lines. A comparison of cAMP levels was performed for ADCY5 wild-type and R418W mutant cells. ADCY5-catalyzed cAMP production was reduced with all three purine derivatives, while the most pronounced effects on cAMP reduction were observed for ADCY5 R418W mutant cells. The gain-of-function ADCY5 R418W mutant is characterized by an increased catalytic activity resulting in elevated cAMP levels that cause kinetic disorders or dyskinesia in patients. Based on our findings in ADCY5 cells, a slow-release formulation of theophylline was administered to a preschool-aged patient with ADCY5-related dyskinesia. A striking improvement of symptoms was observed, outperforming the effects of caffeine that had previously been administered to the same patient. We suggest considering theophylline as an alternative therapeutic option to treat ADCY5-related dyskinesia in patients.


Assuntos
Discinesias , Teofilina , Humanos , Pré-Escolar , Cafeína , Inibidores de Fosfodiesterase , Broncodilatadores , Diuréticos , Vasodilatadores , Agitação Psicomotora
4.
Angew Chem Int Ed Engl ; 61(46): e202205726, 2022 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-36115020

RESUMO

α-Synuclein (α-syn) is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS), fibrillation, and forms insoluble intracellular Lewy bodies in neurons, which are the hallmark of Parkinson's Disease (PD). Neurotoxicity precedes the formation of aggregates and might be related to α-syn LLPS. The molecular mechanisms underlying the early stages of LLPS are still elusive. To obtain structural insights into α-syn upon LLPS, we take advantage of cross-linking/mass spectrometry (XL-MS) and introduce an innovative approach, termed COMPASS (COMPetitive PAiring StatisticS). In this work, we show that the conformational ensemble of α-syn shifts from a "hairpin-like" structure towards more "elongated" conformational states upon LLPS. We obtain insights into the critical initial stages of LLPS and establish a novel mass spectrometry-based approach that will aid to solve open questions in LLPS structural biology.


Assuntos
Proteínas Intrinsicamente Desordenadas , Doença de Parkinson , Humanos , alfa-Sinucleína/química , Doença de Parkinson/metabolismo , Proteínas Intrinsicamente Desordenadas/química , Neurônios/metabolismo , Conformação Molecular
5.
Anal Bioanal Chem ; 413(26): 6503-6511, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34427712

RESUMO

We describe a rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the direct detection and quantitation of SARS-CoV-2 nucleoprotein in gargle solutions and saliva. The method is based on a multiple-reaction monitoring (MRM) mass spectrometry approach with a total cycle time of 5 min per analysis and allows the detection and accurate quantitation of SARS-CoV-2 nucleoprotein as low as 500 amol/µL. We improved the sample preparation protocol of our recent piloting SARS-CoV-2 LC-MS study regarding sensitivity, reproducibility, and compatibility with a complementary reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of the same sample. The aim of this work is to promote diagnostic tools that allow identifying and monitoring SARS-CoV-2 infections by LC-MS/MS methods in a routine clinical environment.


Assuntos
Teste para COVID-19/métodos , COVID-19/diagnóstico , SARS-CoV-2/isolamento & purificação , Saliva/virologia , Espectrometria de Massas em Tandem/métodos , Teste para COVID-19/economia , Cromatografia Líquida/economia , Cromatografia Líquida/métodos , Proteínas do Nucleocapsídeo de Coronavírus/análise , Proteínas do Nucleocapsídeo de Coronavírus/isolamento & purificação , Humanos , Limite de Detecção , Fosfoproteínas/análise , Fosfoproteínas/isolamento & purificação , Reprodutibilidade dos Testes , Manejo de Espécimes , Espectrometria de Massas em Tandem/economia , Fatores de Tempo
6.
Anal Chem ; 93(33): 11442-11450, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34375526

RESUMO

The combination of cross-linking/mass spectrometry (XL-MS) and ion mobility is still underexplored for conducting protein conformational and protein-protein interaction studies. We present a method for analyzing cross-linking mixtures on a timsTOF Pro mass spectrometer that allows separating ions based on their gas-phase mobilities. Cross-linking was performed with three urea-based MS-cleavable cross-linkers that deliver distinct fragmentation patterns for cross-linked species upon collisional activation. The discrimination of cross-linked species from non-cross-linked peptides was readily performed based on their collisional cross sections. We demonstrate the general feasibility of our combined XL-MS/ion mobility approach for three protein systems of increasing complexity: (i) bovine serum albumin (BSA), (ii) Escherichia coli ribosome, and (iii) HEK293T cell nuclear lysates. We identified a total of 623 unique cross-linking sites for BSA, 670 for the E. coli ribosome, and 1623 unique cross-links for nuclear lysates, corresponding to 1088 intra- and 535 interprotein interactions and yielding 564 distinct protein-protein interactions. Our results underline the strength of combining XL-MS with ion mobility not only for deriving three-dimensional (3D) structures of single proteins but also for performing system-wide protein interaction studies.


Assuntos
Escherichia coli , Proteômica , Reagentes de Ligações Cruzadas , Células HEK293 , Humanos , Íons , Espectrometria de Massas , Soroalbumina Bovina
7.
J Mol Biol ; 433(10): 166947, 2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33744315

RESUMO

The rod-outer-segment guanylyl cyclase 1 (ROS-GC1) is a key transmembrane protein for retinal phototransduction. Mutations of ROS-GC1 correlate with different retinal diseases that often lead to blindness. No structural data are available for ROS-GC1 so far. We performed a 3D-structural analysis of native ROS-GC1 from bovine retina by cross-linking/mass spectrometry (XL-MS) and computational modeling. Absolute quantification and activity measurements of native ROS-GC1 were performed by MS-based assays directly in bovine retina samples. Our data present the first 3D-structural analysis of active, full-length ROS-GC1 derived from bovine retina. We propose a novel domain organization for the intracellular domain ROS-GC1. Our XL-MS data of native ROS-GC1 from rod-outer-segment preparations of bovine retina agree with a dimeric architecture. Our integrated approach can serve as a blueprint for conducting 3D-structural studies of membrane proteins in their native environment.


Assuntos
GMP Cíclico/química , Guanilato Ciclase/química , Peptídeos/metabolismo , Receptores de Superfície Celular/química , Segmento Externo da Célula Bastonete/química , Motivos de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Clonagem Molecular , Reagentes de Ligações Cruzadas/química , GMP Cíclico/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Guanilato Ciclase/genética , Guanilato Ciclase/metabolismo , Células HEK293 , Humanos , Espectrometria de Massas/métodos , Modelos Moleculares , Peptídeos/síntese química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Succinimidas/química
8.
J Mass Spectrom ; 50(12): 1386-92, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26634972

RESUMO

Phospholipids are major components of cell membranes and lipoprotein complexes. They are prone to oxidation by endogenous and exogenous reactive oxygen species yielding a large variety of modified lipids including small aliphatic and phospholipid bound aldehydes and ketones. These carbonyls are strong electrophiles that can modify proteins and, thereby, alter their structures and functions triggering various pathophysiological conditions. The analysis of lipid-protein adducts by liquid chromatography-MS is challenged by their mixed chemical nature (polar peptide and hydrophobic lipid), low abundance in biological samples, and formation of multiple isomers. Thus, we investigated traveling wave ion mobility mass spectrometry (TWIMS) to analyze lipid-peptide adducts generated by incubating model peptides corresponding to the amphipathic ß1 sheet sequence of apolipoprotein B-100 with 1-palmitoyl-2-(oxo-nonanoyl)-sn-glycerophosphatidylcholine (PONPC). The complex mixture of peptides, lipids, and peptide-lipid adducts was separated by TWIMS, which was especially important for the identification of two mono-PONPC-peptide isomers containing Schiff bases at different lysine residues. Moreover, TWIMS separated structural conformers of one peptide-lipid adduct possessing most likely different orientations of the hydrophobic sn-1 fatty acyl residue and head group of PONPC, relative to the peptide backbone.


Assuntos
Lipídeos/química , Espectrometria de Massas/métodos , Modelos Químicos , Peptídeos/química , Apolipoproteína B-100/química , Isomerismo , Fosfatidilcolinas/química
9.
Anal Bioanal Chem ; 395(8): 2443-56, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19876624

RESUMO

As more and more alternative treatments become available for breast carcinoma, there is a need to stratify patients and individual molecular information seems to be suitable for this purpose. In this study, we applied label-free protein quantitation by nanoscale LC-MS and investigated whether this approach could be used for defining a proteome signature for invasive ductal breast carcinoma. Tissue samples from healthy breast and tumor were collected from three patients. Protein identifications were based on LC-MS peptide fragmentation data which were obtained simultaneously to the quantitative information. Hereby, an invasive ductal breast carcinoma proteome signature was generated which contains 60 protein entries. The on-column concentrations for osteoinductive factor, vimentin, GAP-DH, and NDKA are provided as examples. These proteins represent distinctive gene ontology groups of differentially expressed proteins and are discussed as risk markers for primary tumor pathogenesis. The developed methodology has been found well applicable in a clinical environment in which standard operating procedures can be kept; a prerequisite for the definition of molecular parameter sets that shall be capable for stratification of patients.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Cromatografia Líquida/métodos , Proteoma/análise , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/patologia , Biologia Computacional , Feminino , Humanos , Técnicas Imunoenzimáticas , Nanotecnologia
10.
FEBS Lett ; 550(1-3): 18-22, 2003 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-12935879

RESUMO

Translocation of folded proteins across biological membranes can be mediated by the so-called 'twin-arginine translocation' (Tat) system. To be translocated, Tat substrates require N-terminal signal sequences which usually contain the eponymous twin-arginine motif. Here we report the first structural analysis of a twin-arginine signal sequence, the signal sequence of the high potential iron-sulfur protein from Allochromatium vinosum. Nuclear magnetic resonance (NMR) analyses of amide proton resonances did not indicate a signal sequence structure. Accordingly, data from H/D exchange matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry showed that the amide protons of the signal sequence exchange rapidly, indicating the absence of secondary structure in the signal sequence up to L29. We conclude that the conserved twin-arginine motif does not form a structure by itself or as a result of intramolecular interactions.


Assuntos
Proteínas de Bactérias/química , Dipeptídeos/química , Proteínas Ferro-Enxofre/química , Complexo de Proteínas do Centro de Reação Fotossintética , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Chromatiaceae/química , Hidrogênio/química , Proteínas Ferro-Enxofre/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Transporte Proteico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos
11.
J Mass Spectrom ; 38(3): 271-6, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12644988

RESUMO

The measurement of deuterium incorporation kinetics using hydrogen/deuterium (H/D) exchange experiments is a valuable tool for the investigation of the conformational dynamics of biomolecules in solution. Experiments consist of two parts when using H/D exchange mass spectrometry to analyse the deuterium incorporation. After deuterium incorporation at high D(2)O concentration, it is necessary to decrease the D(2)O concentration before the mass analysis to avoid deuterium incorporation under artificial conditions of mass spectrometric preparation and measurement. A low D(2)O concentration, however, leads to back-exchange of incorporated deuterons during mass analysis. This back-exchange is one of the major problems in H/D exchange mass spectrometry and must be reduced as much as possible. In the past, techniques using electrospray ionization (ESI) had the lowest back-exchange values possible in H/D exchange mass spectrometry. Methods for the measurement of H/D exchange by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) that have been developed since 1998 have some significant advantages, but they could not achieve the back-exchange minima of ESI methods. Here, we present a protocol for H/D exchange MALDI-MS which allows for greater minimization of back-exchange compared with H/D exchange ESI-MS under similar conditions.


Assuntos
Deutério/química , Hidrogênio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ciclofilinas/química , Ciclosporina/química , Humanos , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Conformação Proteica
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