RESUMO
Alzheimer's disease is the most common form of dementia and the generation of oligomeric species of amyloid-ß is causal to the initiation and progression of it. Amyloid-ß oligomers bind to the N-terminus of plasma membrane-bound cellular prion protein (PrP(C)) initiating a series of events leading to synaptic degeneration. Composition of bound amyloid-ß oligomers, binding regions within PrP(C), binding affinities and modifiers of this interaction have been almost exclusively studied in cell culture or murine models of Alzheimer's disease and our knowledge on PrP(C)-amyloid-ß interaction in patients with Alzheimer's disease is limited regarding occurrence, binding regions in PrP(C), and size of bound amyloid-ß oligomers. Here we employed a PrP(C)-amyloid-ß binding assay and size exclusion chromatography on neuropathologically characterized Alzheimer's disease and non-demented control brains (n = 15, seven female, eight male, average age: 79.2 years for Alzheimer's disease and n = 10, three female, seven male, average age: 66.4 years for controls) to investigate amyloid-ß-PrP(C) interaction. PrP(C)-amyloid-ß binding always occurred in Alzheimer's disease brains and was never detected in non-demented controls. Neither expression level of PrP(C) nor known genetic modifiers of Alzheimer's disease, such as the PrP(C) codon 129 polymorphism, influenced this interaction. In Alzheimer's disease brains, binding of amyloid-ß to PrP(C) occurred via the PrP(C) N-terminus. For synthetic amyloid-ß42, small oligomeric species showed prominent binding to PrP(C), whereas in Alzheimer's disease brains larger protein assemblies containing amyloid-ß42 bound efficiently to PrP(C). These data confirm Alzheimer's disease specificity of binding of amyloid-ß to PrP(C) via its N-terminus in a large cohort of Alzheimer's disease/control brains. Differences in sizes of separated protein fractions between synthetic and brain-derived amyloid-ß binding to PrP(C) suggest that larger assemblies of amyloid-ß or additional non-amyloid-ß components may play a role in binding of amyloid-ß42 to PrP(C) in Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas PrPC/metabolismo , Córtex Pré-Frontal/metabolismo , Ligação Proteica , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/química , Cromatografia em Gel , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético/genética , Proteínas PrPC/química , Proteínas PrPC/genética , Córtex Pré-Frontal/química , Córtex Pré-Frontal/patologia , Proteínas Priônicas , Príons/genética , Príons/metabolismoRESUMO
Podocytes are the key cells affected in nephrotic glomerular kidney diseases, and they respond uniformly to injury with cytoskeletal rearrangement. In nephrotic diseases, such as membranous nephropathy and FSGS, persistent injury often leads to irreversible structural damage, whereas in minimal change disease, structural alterations are mostly transient. The factors leading to persistent podocyte injury are currently unknown. Proteolysis is an irreversible process and could trigger persistent podocyte injury through degradation of podocyte-specific proteins. We, therefore, analyzed the expression and functional consequence of the two most prominent proteolytic systems, the ubiquitin proteasome system (UPS) and the autophagosomal/lysosomal system, in persistent and transient podocyte injuries. We show that differential upregulation of both proteolytic systems occurs in persistent human and rodent podocyte injury. The expression of specific UPS proteins in podocytes differentiated children with minimal change disease from children with FSGS and correlated with poor clinical outcome. Degradation of the podocyte-specific protein α-actinin-4 by the UPS depended on oxidative modification in membranous nephropathy. Notably, the UPS was overwhelmed in podocytes during experimental glomerular disease, resulting in abnormal protein accumulation and compensatory upregulation of the autophagosomal/lysosomal system. Accordingly, inhibition of both proteolytic systems enhanced proteinuria in persistent nephrotic disease. This study identifies altered proteolysis as a feature of persistent podocyte injury. In the future, specific UPS proteins may serve as new biomarkers or therapeutic targets in persistent nephrotic syndrome.
Assuntos
Podócitos/metabolismo , Podócitos/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteinúria/metabolismo , Proteinúria/patologia , Ubiquitina/metabolismo , Actinina/genética , Actinina/metabolismo , Animais , Autofagia/fisiologia , Linhagem Celular Transformada , Modelos Animais de Doenças , Humanos , Glomérulos Renais/metabolismo , Glomérulos Renais/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Complexo de Endopeptidases do Proteassoma/genética , Proteinúria/genética , Ratos Wistar , Transcriptoma , Ubiquitina/genética , Regulação para Cima/fisiologiaRESUMO
The influence of packing process parameters (packing pressure, application of ultrasound) and the stationary phase particle size (3.5 and 5 µm) on the chromatographic performance of HPLC/MS chips was systematically investigated for proteomic samples. First, reproducibility and detection limits of the separation were evaluated with a low-complexity sample of tryptic BSA peptides. The influence of adsorbent packing quality on protein identification was then tested with a typical proteomics sample of high complexity, a human plasma protein fraction (Cohn fraction IV-4). All HPLC/MS chips provided highly reproducible separations of these proteomic samples, but improved packing conditions and smaller particle sizes resulted in chromatograms with narrower peaks and correspondingly higher signal intensities. Improved separation performance increased the peak capacity, the number of identified peptides, and thus the sequence coverage in the proteomic samples, particularly for low sample amounts.
Assuntos
Cromatografia Líquida de Alta Pressão , Procedimentos Analíticos em Microchip , Proteômica , Espectrometria de Massas em Tandem , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Dispositivos Lab-On-A-Chip , Procedimentos Analíticos em Microchip/métodos , Proteômica/instrumentação , Proteômica/métodos , Espectrometria de Massas em Tandem/instrumentação , Espectrometria de Massas em Tandem/métodos , Tripsina/metabolismoRESUMO
The Pseudechis colletti and Pseudechis butleri venoms were analyzed by 1-D gel electrophoresis, followed by mass spectrometric analysis of tryptic peptides obtained from the protein bands. Both venoms contain highly potent pharmacologically active components, which were assigned to the following protein families: basic and acidic phospholipases A2 (PLA2s), L-amino acid oxidases (LAAOs), P-III metalloproteinases (P-III SVMPs), 5'- nucleotidases (5'-NTDs), cysteine-rich secretory proteins (CRISPs), venom nerve growth factors (VNGFs) and post-synaptic neurotoxins. Considerable predominance of PLA2s over other toxins is a characteristic feature of both venoms. The major differences in the venom compositions are the higher concentration of SVMPs and CRISPs in the P. butleri venom, as well as the presence of post-synaptic neurotoxins. Furthermore, the analysis revealed a high concentration of proteins with myotoxic, coagulopathic and apoptotic activities. PLA2s are responsible for the myotoxic and anticoagulant effects observed in patients after envenomation (4). The other protein families, encountered in the two venoms, probably contribute to the major symptoms described for these venoms. These results explain the observed clinical effects of the black snake envenomation. The analyzed venoms contain group P-III metalloproteinases of medical importance with the potency to be used for diagnostic purposes of von Willebrand factor (vWF) disease, for regulation of vWF in thrombosis and haemostasis, for studying the function of the complement system in host defense and in the pathogenesis of diseases. Comparison of venomic data showed similarities in the major venom components of snakes from the genus Pseudechis, resulting in common clinical effects of envenomation, and demonstrating close relationships between venom toxins of Elapidae snakes.
Assuntos
Peptídeos/análise , Proteoma/análise , Venenos de Serpentes/metabolismo , Serpentes/metabolismo , Aminoácido Oxirredutases/análise , Aminoácido Oxirredutases/metabolismo , Animais , Austrália , Eletroforese em Gel de Poliacrilamida , Metaloendopeptidases/análise , Metaloendopeptidases/metabolismo , Fatores de Crescimento Neural/análise , Fatores de Crescimento Neural/metabolismo , Peptídeos/metabolismo , Fosfolipases A2/análise , Fosfolipases A2/metabolismo , Espectrometria de Massas em TandemRESUMO
The eastern brown snake is the predominant cause of snakebites in mainland Australia. Its venom induces defibrination coagulopathy, renal failure and microangiopathic hemolytic anemia. Cardiovascular collapse has been described as an early cause of death in patients, but, so far, the mechanisms involved have not been fully identified. In the present work, we analysed the venome of Pseudonaja textilis by combining high throughput proteomics and transcriptomics, aiming to further characterize the components of this venom. The combination of these techniques in the analysis and identification of toxins, venom proteins and putative toxins allowed the sequence description and the identification of the following: prothrombinase coagulation factors, neurotoxic textilotoxin phospholipase A2 (PLA2) subunits and "acidic PLA2", three-finger toxins (3FTx) and the Kunitz-type protease inhibitor textilinin, venom metalloproteinase, C-type lectins, cysteine rich secretory proteins, calreticulin, dipeptidase 2, as well as evidences of Heloderma lizard peptides. Deep data-mining analysis revealed the secretion of a new transcript variant of venom coagulation factor 5a and the existence of a splicing variant of PLA2 modifying the UTR and signal peptide from a same mature protein. The transcriptome revealed the diversity of transcripts and mutations, and also indicates that splicing variants can be an important source of toxin variation.
Assuntos
Venenos Elapídicos/química , Elapidae , Proteínas de Répteis/química , Processamento Alternativo , Animais , Austrália , Perfilação da Expressão Gênica , Proteômica , Proteínas de Répteis/isolamento & purificação , Análise de Sequência de ProteínaRESUMO
The investigation of venoms has many clinical, pharmacological, ecological and evolutionary outcomes. The Crotalus spp. venom can cause hemorrhage, neurotoxicity, myotoxicity, coagulopathy and hypotension. Although neurotoxicity and hemorrhage usually does not occur for the same species, the rare Venezuelan species Crotalus vegrandis presents both characteristic. Different from the other species it has a restricted ecological niche and geographical distribution. Nevertheless, it has a raising medical importance as this rattlesnake population is increasing. Few works describe its neurotoxic and hemorrhagic features, but other toxins might play an important role in envenomation. We combined proteomic methods to identify for the first time the main components of it venom: 2D SDS-PAGE and gel-filtration chromatography for protein mixture decomplexation; LC-MS(2) of low molecular mass fractions and tryptic peptides; bioinformatic identification of toxin families and specific protein species based on unique peptide analysis and sequence database enriched with species-specific venom gland transcripts; and finally polyclonal anti-crotamine Western-blotting. Our results point to a broad arsenal of toxins in C. vegrandis venom: PIII and PII metalloproteases, crotoxin subunits, other phospholipases, isoforms of serine proteases and lectins, l-amino-acid oxidase, nerve growth factor, as well as other less abundant toxins.
Assuntos
Venenos de Crotalídeos/química , Crotalus/metabolismo , Metaloproteases/química , Proteômica , Proteínas de Répteis/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Espectrometria de Massas , Metaloproteases/isolamento & purificação , Dados de Sequência Molecular , Proteínas de Répteis/isolamento & purificaçãoRESUMO
The human serine protease high temperature requirement A1 (HTRA1) is highly expressed in the placental tissue, especially in the last trimester of gestation. This suggests that HTRA1 is involved in placental formation and function. With the aim of a better understanding of the role of HTRA1 in the placenta, candidate substrates were screened in a placenta protein extract using a gel-based mass spectrometric approach. Protease inhibitor alpha-1-antitrypsin, actin cytoplasmic 1, tropomyosin beta chain and ten further proteins were identified as candidate substrates of HTRA1. Among the identified candidate substrates, alpha-1-antitrypsin (A1AT) was considered to be of particular interest because of its important role as protease inhibitor. For investigation of alpha-1-antitrypsin as substrate of HTRA1 synthetic peptides covering parts of the sequence of alpha-1-antitrypsin were incubated with HTRA1. By mass spectrometry a specific cleavage site was identified after met-382 (AIPM382↓383SIPP) within the reactive centre loop of alpha-1-antitrypsin, resulting in a C-terminal peptide comprising 36 amino acids. Proteolytic removal of this peptide from alpha-1-antitrypsin results in a loss of its inhibitor function. Beside placental alpha-1-antitrypsin the circulating form in human plasma was also significantly degraded by HTRA1. Taken together, our data suggest a link between the candidate substrates alpha-1-antitrypsin and the function of HTRA1 in the placenta in the syncytiotrophoblast, the cell layer attending to maternal blood in the villous tree of the human placenta. Data deposition: Mass spectrometry (MS) data have been deposited to the ProteomeXchange with identifier PXD000473.
Assuntos
Placenta/metabolismo , Serina Endopeptidases/metabolismo , alfa 1-Antitripsina/metabolismo , Sequência de Aminoácidos , Animais , Feminino , Serina Peptidase 1 de Requerimento de Alta Temperatura A , Humanos , Espectrometria de Massas , Dados de Sequência Molecular , Placenta/citologia , Gravidez , Ligação Proteica , Proteólise , Inibidores de Serina Proteinase/farmacologia , alfa 1-Antitripsina/químicaRESUMO
The Australian Elapidae spotted black snake Pseudechis guttatus venom proteome composition was analyzed by high throughput mass spectrometry. The crude venom proteins were decomplexed by 2D-PAGE and in-gel digestion peptides from 66 spot samples and analyzed by tandem mass spectrometry-LC-ESI-ion trap. Protein identification was performed combining PEAKS studio 7.0 and Mascot software. The analysis identified l-amino-acid oxidases, phospholipases A2, metalloproteases, nerve growth factors and ecto-5'-nucleotidases, and for the first time in this venom the components cysteine-rich secretory proteins similar to pseudechetoxin, phospholipase B and transferrin-like protein. The envenomation symptoms are in agreement with the identified components, but the present limitations of database information might impair the detection of toxin families, protein species and still unknown toxins. From the qualitative point of view, the similarity of this venom with the ones from other Pseudechis species could be assigned to recent speciation events. BIOLOGICAL SIGNIFICANCE: Studies on the proteome of Australian Elapidae (Ancanthophiinae) are quite rare. In the present work we performed, using classic proteomic methods, a qualitative and partial analysis of the proteic components of Pseudechis guttatus venom. Although previous studies contributed to the knowledge of the major components of this venom, our study revealed some yet undescribed protein species, as well as new toxins, such as CRiSPs, phospholipase B, transferrin-like protein and ecto 5'-nucleotidase.
Assuntos
Evolução Biológica , Venenos Elapídicos/química , Venenos Elapídicos/genética , Elapidae/genética , Evolução Molecular , Família Multigênica/genética , Proteoma/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Elapidae/classificação , Variação Genética , Dados de Sequência Molecular , Análise de Sequência de Proteína/métodos , Especificidade da EspécieRESUMO
Elapid snake venom is a highly valuable, but till now mainly unexplored, source of pharmacologically important peptides. We analyzed the peptide fractions with molecular masses up to 10 kDa of two elapid snake venoms-that of the African cobra, N. m. mossambica (genus Naja), and the Peninsula tiger snake, N. scutatus, from Kangaroo Island (genus Notechis). A combination of chromatographic methods was used to isolate the peptides, which were characterized by combining complimentary mass spectrometric techniques. Comparative analysis of the peptide compositions of two venoms showed specificity at the genus level. Three-finger (3-F) cytotoxins, bradykinin-potentiating peptides (BPPs) and a bradykinin inhibitor were isolated from the Naja venom. 3-F neurotoxins, Kunitz/basic pancreatic trypsin inhibitor (BPTI)-type inhibitors and a natriuretic peptide were identified in the N. venom. The inhibiting activity of the peptides was confirmed in vitro with a selected array of proteases. Cytotoxin 1 (P01467) from the Naja venom might be involved in the disturbance of cellular processes by inhibiting the cell 20S-proteasome. A high degree of similarity between BPPs from elapid and viperid snake venoms was observed, suggesting that these molecules play a key role in snake venoms and also indicating that these peptides were recruited into the snake venom prior to the evolutionary divergence of the snakes.
Assuntos
Venenos Elapídicos/química , Elapidae , Peptídeos/isolamento & purificação , Inibidores de Proteases/isolamento & purificação , Sequência de Aminoácidos , Animais , Bradicinina/antagonistas & inibidores , Cromatografia em Gel , Cromatografia Líquida , Quimotripsina/antagonistas & inibidores , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Peptídeos/farmacologia , Peptidil Dipeptidase A/metabolismo , Inibidores de Proteases/farmacologia , Alinhamento de Sequência , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Subtilisina/antagonistas & inibidores , Tripsina/metabolismoRESUMO
In mammalian species, except humans, N-terminal processing of the precursor peptide angiotensin I (ANG-1-10) into ANG-2-10 or ANG-3-10 was reported. Here we hypothesize that aminopeptidase-generated angiotensins bearing the same C-terminus as ANG-1-10 are also present in humans. We demonstrate the time dependent generation of ANG-2-10, ANG-3-10, ANG-4-10, ANG-5-10 and ANG-6-10 from the precursor ANG-1-10 by human plasma proteins. The endogenous presence of ANG-4-10, ANG-5-10 and ANG-6-10 in human plasma was confirmed by an immuno-fluorescence assay. Generation of ANG-2-10, ANG-3-10 and ANG-4-10 from ANG-1-10 by immobilized human plasma proteins was sensitive to the cysteine/serine protease inhibitor antipain. The metal ion chelator EDTA inhibited Ang-6-10-generation. Incubation of the substrates ANG-3-10, ANG-4-10 and ANG-5-10 with recombinant aminopeptidase N (APN) resulted in a successive N-terminal processing, finally releasing ANG-6-10 as a stable end product, demonstrating a high similarity concerning the processing pattern of the angiotensin peptides compared to the angiotensin generating activity in plasma. Recombinant ACE-1 hydrolyzed the peptides ANG-2-10, ANG-3-10, ANG-4-10 and ANG-5-10 into ANG-2-8, ANG-3-8, ANG-4-8 and ANG-5-8. Since ANG-2-10 was processed into ANG-2-8, ANG-4-8 and ANG-5-8 by plasma proteases the angiotensin peptides bearing the same C-terminus as ANG-1-10 likely have a precursor function in human plasma. Our results confirm the hypothesis of aminopeptidase mediated processing of ANG-1-10 in humans. We show the existence of an aminopeptidase mediated pathway in humans that bypasses the known ANG-1-8-carboxypeptidase pathway. This expands the knowledge about the known human renin angiotensin system, showing how efficiently the precursor ANG-1-10 is used by nature.
Assuntos
Angiotensina I/sangue , Proteólise , Sequência de Aminoácidos , Angiotensina I/química , Angiotensina I/isolamento & purificação , Angiotensinas/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Antígenos CD13/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Proteínas Imobilizadas/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Peptidil Dipeptidase A/metabolismo , Inibidores de Proteases/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
BACKGROUND: Proteases play an essential part in a variety of biological processes. Besides their importance under healthy conditions they are also known to have a crucial role in complex diseases like cancer. In recent years, it has been shown that not only the fragments produced by proteases but also their dynamics, especially ex vivo, can serve as biomarkers. But so far, only a few approaches were taken to explicitly model the dynamics of proteolysis in the context of mass spectrometry. RESULTS: We introduce a new concept to model proteolytic processes, the degradation graph. The degradation graph is an extension of the cleavage graph, a data structure to reconstruct and visualize the proteolytic process. In contrast to previous approaches we extended the model to incorporate endoproteolytic processes and present a method to construct a degradation graph from mass spectrometry time series data. Based on a degradation graph and the intensities extracted from the mass spectra it is possible to estimate reaction rates of the underlying processes. We further suggest a score to rate different degradation graphs in their ability to explain the observed data. This score is used in an iterative heuristic to improve the structure of the initially constructed degradation graph. CONCLUSION: We show that the proposed method is able to recover all degraded and generated peptides, the underlying reactions, and the reaction rates of proteolytic processes based on mass spectrometry time series data. We use simulated and real data to demonstrate that a given process can be reconstructed even in the presence of extensive noise, isobaric signals and false identifications. While the model is currently only validated on peptide data it is also applicable to proteins, as long as the necessary time series data can be produced.
Assuntos
Bases de Dados de Proteínas , Espectrometria de Massas/métodos , Modelos Biológicos , Proteólise , Simulação por Computador , Fibrinopeptídeo A/química , Fibrinopeptídeo A/metabolismo , Humanos , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Microglobulina beta-2/química , Microglobulina beta-2/metabolismoRESUMO
Liquid chromatography is often the method of choice for the analysis of proteins in their native state. Nevertheless compared to two-dimensional electrophoresis, the resolution of common chromatographic techniques is low. Liquid chromatography in the displacement mode has previously been shown to offer higher resolution and to elute proteins in the high concentrations. In this study we compared to what extend displacement mode was a suitable alternative to gradient mode for the separation of a complex protein mixture using anion-exchange displacement chromatography and if it is therefore helpful for proteomic investigations. Hence we analyzed the qualitative protein composition of each fraction by tryptic digestion of the proteins, analysis of the tryptic peptides by liquid chromatography coupled to mass spectrometry followed by data base analysis and by measuring the elution profiles of 22 selected proteins with selected reaction monitoring mass spectrometry. In the fractions of displacement mode a significantly higher number of identified proteins (51 versus 16) was yielded in comparison to gradient mode. The resolution of displacement chromatography was slightly lower than of gradient chromatography for many but not for all proteins. The selectivities of displacement mode and gradient mode are very different. In conclusion displacement chromatography is a well suited alternative for top-down proteomic approaches which start with separating intact proteins first prior to mass spectrometric analysis of intact or digested proteins. The significant orthogonality of both modes may be used in the future for combining them in multidimensional fractionation procedures.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Cromatografia por Troca Iônica/métodos , Espectrometria de Massas/métodos , Ânions/química , Proteínas Sanguíneas/química , Condutividade Elétrica , Humanos , Espectrofotometria UltravioletaRESUMO
Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Proteômica , Talidomida/farmacologia , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Desenvolvimento Embrionário/genética , Células-Tronco Embrionárias/citologia , Extremidades/embriologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Coração/efeitos dos fármacos , Coração/embriologia , Humanos , Cinética , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/genéticaRESUMO
Defining membrane proteomes is fundamental to understand the role of membrane proteins in biological processes and to find new targets for drug development. Usually multidimensional chromatography using step or gradient elution is applied for the separation of tryptic peptides of membrane proteins prior to their mass spectrometric analysis. Displacement chromatography (DC) offers several advantages that are helpful for proteome analysis. However, DC has so far been applied for proteomic investigations only in few cases. In this study we therefore applied DC in a multidimensional LC-MS approach for the separation and identification of membrane proteins located in cholesterol-enriched membrane microdomains (lipid rafts) obtained from rat kidney by density gradient centrifugation. The tryptic peptides were separated on a cation-exchange column in the displacement mode with spermine used as displacer. Fractions obtained from DC were analyzed using an HPLC-chip system coupled to an electrospray-ionization ion-trap mass spectrometer. This procedure yielded more than 400 highly significant peptide spectrum matches and led to the identification of more than 140 reliable protein hits within an established rat kidney lipid raft proteome. The majority of identified proteins were membrane proteins. In sum, our results demonstrate that DC is a suitable alternative to gradient elution separations for the identification of proteins via a multidimensional LC-MS approach.
Assuntos
Cromatografia Líquida/métodos , Microdomínios da Membrana/química , Proteoma/análise , Sequência de Aminoácidos , Animais , Resinas de Troca de Cátion , Immunoblotting , Rim/metabolismo , Masculino , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma/química , Ratos , Ratos WistarRESUMO
It was the aim of this study to compare the performance of displacement chromatography with gradient elution chromatography both applied as the cation-exchange separation step for a proteome analysis in a bottom-up approach using multidimensional chromatography for the separation of tryptic peptides prior to their mass spectrometric analysis. The tryptic digest of the human Cohn fraction IV-4 served as a sample. For both chromatography modes commonly used operating parameters were chosen thus ensuring optimal separation results of equal sample amounts for each mode. All resulting fractions were analyzed with an HPLC-chip-LC-MS system. The eluate of the HPLC-chip column was ionized by electrospray ionization (ESI) and analyzed with an ion-trap mass spectrometer. For guaranteeing high confidence concerning the identity of the peptides, the mass spectrometric data were processed by different bioinformatic tools applying stringent criteria. By the displacement approach the total amount of identified proteins (78) was significantly higher than in the gradient mode (58). The results showed that displacement chromatography is a well suited alternative in comparison to gradient elution separation for analysis of proteomes via the bottom-up approach applying multidimensional chromatography, especially in those cases when larger quantities of proteins are available.