RESUMO
We report a loss-less two-dimensional (2D) separation platform that integrated capillary zone electrophoresis (CZE) fractionation and nanoRPLC-ESI-MS/MS for a comprehensive proteomics analysis of a submicrogram sample. Protein digest was injected into the linear polyacrylamide-coated capillary, followed by CZE separation. The schemes for collecting the fractions were carefully optimized to maximize the protein coverage. The peptide fractions were directly eluted into the autosampler insert vials, followed by the nanoRPLC-ESI-MS/MS analysis without lyophilization and redissolution, thus dramatically minimizing sample loss and potential contamination. The integrated platform generated 30,845 unique peptides and 5231 protein groups from 500 ng of a HeLa protein digest within 11.5 h (90 min CZE fractionation plus 10 h LC-MS analysis). Finally, the developed platform was used to analyze the protein digest prepared by the MICROFASP method with 1 µg of cell lysate as the starting material. Three thousand seven hundred ninety-six (N = 2, RSD = 4.95%) protein groups and 20,577 (N = 2, RSD = 7.89%) peptides were identified from only 200 ng of the resulted tryptic digest within 5.5 h. The results indicated that the combination of the MICROFASP method and the developed CZE/nanoRPLC-MS/MS 2D separation platform enabled comprehensive proteome profiling of a submicrogram biological sample. Data are available via ProteomeXchange with the identifier PXD052735.
Assuntos
Eletroforese Capilar , Proteômica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Humanos , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Eletroforese Capilar/métodos , Células HeLa , Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/análise , Peptídeos/química , Peptídeos/isolamento & purificação , Proteoma/análise , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas/química , Fracionamento Químico/métodosRESUMO
Anthocyanins, as the most critical water-soluble pigments in nature, are widely present in roots, stems, leaves, flowers, fruits, and fruit peels. Many studies have indicated that anthocyanins exhibit various biological activities including antioxidant, anti-inflammatory, anti-tumor, hypoglycemic, vision protection, and anti-aging. Hence, anthocyanins are widely used in food, medicine, and cosmetics. The green and efficient extraction and purification of anthocyanins are an important prerequisite for their further development and utilization. However, the poor stability and low bioavailability of anthocyanins limit their application. Protein, one of the three essential nutrients for the human body, has good biocompatibility and biodegradability. Proteins are commonly used in food processing, but their functional properties need to be improved. Notably, anthocyanins can interact with proteins through covalent and non-covalent means during food processing, which can effectively improve the stability of anthocyanins and enhance their bioavailability. Moreover, the interactions between proteins and anthocyanins can also improve the functional characteristics and enhance the nutritional quality of proteins. Hence, this article systematically reviews the extraction and purification methods for anthocyanins. Moreover, this review also systematically summarizes the effect of the interactions between anthocyanins and proteins on the bioavailability of anthocyanins and their impact on protein properties. Furthermore, we also introduce the application of the interaction between anthocyanins and proteins. The findings can provide a theoretical reference for the application of anthocyanins and proteins in food deep processing.
Assuntos
Antocianinas , Antocianinas/química , Antocianinas/isolamento & purificação , Humanos , Proteínas/isolamento & purificação , Proteínas/química , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Disponibilidade Biológica , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificaçãoRESUMO
While nickel-nitrilotriacetic acid (Ni-NTA) has greatly advanced recombinant protein purification, its limitations, including nonspecific binding and partial purification for certain proteins, highlight the necessity for additional purification such as size exclusion and ion exchange chromatography. However, specialized equipment such as FPLC is typically needed but not often available in many laboratories. Here, we show a novel method utilizing polyphosphate (polyP) for purifying proteins with histidine repeats via non-covalent interactions. Our study demonstrates that immobilized polyP efficiently binds to histidine-tagged proteins across a pH range of 5.5-7.5, maintaining binding efficacy even in the presence of reducing agent DTT and chelating agent EDTA. We carried out experiments of purifying various proteins from cell lysates and fractions post-Ni-NTA. Our results demonstrate that polyP resin is capable of further purification post-Ni-NTA without the need for specialized equipment and without compromising protein activity. This cost-effective and convenient method offers a viable approach as a complementary approach to Ni-NTA.
Assuntos
Histidina , Polifosfatos , Histidina/química , Polifosfatos/química , Polifosfatos/metabolismo , Ácido Nitrilotriacético/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Humanos , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
Shrimp processing generates substantial waste, which is rich in valuable components such as polysaccharides, proteins, carotenoids, and fatty acids. This review provides a comprehensive overview of the valorization of shrimp waste, mainly shrimp shells, focusing on extraction methods, bioactivities, and potential applications of these bioactive compounds. Various extraction techniques, including chemical extraction, microbial fermentation, enzyme-assisted extraction, microwave-assisted extraction, ultrasound-assisted extraction, and pressurized techniques are discussed, highlighting their efficacy in isolating polysaccharides, proteins, carotenoids, and fatty acids from shrimp waste. Additionally, the bioactivities associated with these compounds, such as antioxidant, antimicrobial, anti-inflammatory, and antitumor properties, among others, are elucidated, underscoring their potential in pharmaceutical, nutraceutical, and cosmeceutical applications. Furthermore, the review explores current and potential utilization avenues for these bioactive compounds, emphasizing the importance of sustainable resource management and circular economy principles in maximizing the value of shrimp waste. Overall, this review paper aims to provide insights into the multifaceted aspects of shrimp waste valorization, offering valuable information for researchers, industries, and policymakers interested in sustainable resource utilization and waste-management strategies.
Assuntos
Penaeidae , Gerenciamento de Resíduos , Animais , Antioxidantes/farmacologia , Antioxidantes/isolamento & purificação , Antioxidantes/química , Carotenoides/farmacologia , Carotenoides/isolamento & purificação , Carotenoides/química , Ácidos Graxos/isolamento & purificação , Ácidos Graxos/química , Ácidos Graxos/farmacologia , Penaeidae/química , Polissacarídeos/farmacologia , Polissacarídeos/isolamento & purificação , Polissacarídeos/química , Proteínas/isolamento & purificação , Gerenciamento de Resíduos/métodos , ResíduosRESUMO
The venom duct transcriptomes and proteomes of the cryptic cone snail species Virroconus ebraeus and Virroconus judaeus were obtained and compared. The most abundant and shared conotoxin precursor superfamilies in both species were M, O1, and O2. Additionally, three new putative conotoxin precursor superfamilies (Virro01-03) with cysteine pattern types VI/VII and XVI were identified. The most expressed conotoxin precursor superfamilies were SF-mi2 and M in V. ebraeus, and Cerm03 and M in V. judaeus. Up to 16 conotoxin precursor superfamilies and hormones were differentially expressed between both species, and clustered into two distinct sets, which could represent adaptations of each species to different diets. Finally, we predicted, with machine learning algorithms, the 3D structure model of selected venom proteins including the differentially expressed Cerm03 and SF-mi2, an insulin type 3, a Gastridium geographus GVIA-like conotoxin, and an ortholog to the Pionoconus magus ω-conotoxin MVIIA (Ziconotide).
Assuntos
Caramujo Conus , Venenos de Moluscos/química , Proteínas/química , Algoritmos , Animais , Aprendizado de Máquina , Proteínas/isolamento & purificação , Proteoma , Especificidade da Espécie , TranscriptomaRESUMO
BACKGROUND: Due to the association of hypermutated colorectal cancer (CRC) with many neo-antigens, poly-neo-epitopes are attractive vaccines. The molecular features of murine CT26 are similar to those of aggressive human CRC. CT26 contains some antigenic mutations, which can provide specific immunotherapy targets. Herein, we aimed to express, and purify the previously designed hexatope containing CT26 neoepitopes, CT26-poly-neoepitopes. METHODS AND RESULTS: In the current study, expression of the CT26-poly-neoepitopes was optimized in three different Escherichia coli strains including BL21 (DE3), Origami (DE3), and SHuffle®. Furthermore, the effect of ethanol on the CT26-poly-neoepitopes expression was investigated. The highest amount of CT26-poly-neoepitopes, which included CT26-poly-neoepitopes with the uncleaved pelB signal sequence and the processed one, was achieved when BL21 containing pET-22 (CT26-poly-neoepitopes) was induced with 0.1 mM IPTG for 48 h at 22 ºC in the presence of 2% ethanol. However, 37 ºC was the optimized induction temperature for expression of the CT26-poly-neoepitopes in the absence of ethanol. To purify the CT26-poly-neoepitopes, Ni-NTA affinity chromatography under denaturing and hybrid conditions were applied. High and satisfactory CT26-poly-neoepitopes purity was achieved by the combined urea and imidazole method. CONCLUSION: The effect of ethanol on expression of the CT26-poly-neoepitopes was temperature-dependent. Furthermore, the pelB-mediated translocation of the CT26-poly-neoepitopes into the periplasm was inefficient. Moreover, higher concentration of imidazole in the washing buffer improved the CT26-poly-neoepitopes purification under hybrid condition. Overall, the immunogenicity of CT26-poly-neoepitopes expressed in BL21 under the optimum condition and purified under hybrid condition can be studied in our future in vivo study.
Assuntos
Engenharia de Proteínas/métodos , Proteínas/isolamento & purificação , Vacinas/biossíntese , Epitopos/genética , Escherichia coli , Humanos , Imunoterapia , Periplasma , Sinais Direcionadores de ProteínasRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Pheretima is a traditional Chinese medicine that could treat various lung diseases such as asthma, pneumonia, and lung cancer effectively; however, limited studies on the use of Pheretima protein in the treatment of lung diseases have been conducted to date. AIM OF THE STUDY: The aim of this study was to explain the antipulmonary fibrosis mechanism of the Pheretima protein and elucidate its possible cell signaling pathways. MATERIAL AND METHODS: Fresh pheretima was freeze-dried to obtain the Pheretima protein. Divide C57BL/6 mice into control and bleomycin (BLM)-induced models, pirfenidone, and Pheretima protein-treatment groups. Three weeks later, they were treated with H&E and Masson's trichrome staining to assess lung injury and fibrosis. Pulmonary fibrosis was assessed using immunohistochemistry (IHC), realtime-PCR (RT-PCR), and western blotting. Inflammation was assessed using the alveolar lavage fluid. RESULTS: Pheretima protein inhibited epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) deposition and reduced inflammation. It also reduced the levels of Smad2/3, pSmad2/3, and transforming growth factor-beta 1 (TGF-ß1). Thus, our results indicate that Pheretima protein can alleviate BLM-induced pulmonary fibrosis in a mouse model. CONCLUSION: Pheretima protein inhibits ECM, EMT, and antiinflammatory markers, which in turn ameliorates BLM-induced pulmonary fibrosis. Preliminary mechanistic studies indicated that Pheretima protein can exert its biological activity by downregulating the TGF-ß1/Smad2/3 pathway.
Assuntos
Fibrose Pulmonar Idiopática/tratamento farmacológico , Inflamação/tratamento farmacológico , Medicina Tradicional Chinesa/métodos , Proteínas/farmacologia , Animais , Anti-Inflamatórios/isolamento & purificação , Anti-Inflamatórios/farmacologia , Bleomicina , Modelos Animais de Doenças , Liofilização , Fibrose Pulmonar Idiopática/fisiopatologia , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligoquetos/química , Proteínas/isolamento & purificação , Piridonas/farmacologia , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Erigeron canadensis has been used in traditional medicine to treat a variety of respiratory diseases, including acute upper and lower respiratory tract infections and cough-related asthma. There is as yet no relevant experimental or clinical study in the scientific literature evaluating the efficacy of plants in these disorders. AIM OF THE STUDY: To investigate the active ingredients in Erigeron canadensis, a complex isolated from flowering parts of a plant was tested for airway defense reflexes, in particular for cough reflexes and airway reactivity. Both were experimentally induced by a chemical irritant that simulated the inflammatory conditions of their formation. MATERIAL AND METHODS: The polyphenolic polysaccharide-protein (PPP) complex was isolated from the flowering parts of Erigeron canadensis by hot alkaline extraction and a multi-stage purification process. The antitussive activity was confirmed as a decrease in the number of citric acid-induced coughs and the bronchodilator effect was verified as a decrease in specific airway resistance (sRaw) in conscious guinea pigs. RESULTS: The dark brown Erigeron complex with a molecular weight of 38,000 g/mol contained phenolics (13.2% wt%), proteins (16.3% wt%), and uronic acids (6.3% wt%). The neutral carbohydrate part of Erigeron consisted mainly of xylose (12.1 wt%), glucose (13.3 wt%), arabinose (24.1 wt%), and galactose (41.0 wt%) residues. Arabinogalactan and 4-OMe-glucuronoxylan have been found to be the major polysaccharides in the Erigeron complex. Using a method of chemically-induced cough reflex and guinea pigs test system the Erigeron complex exhibited statistically significant, the dose-dependent antitussive activity, which was similar to that of the centrally-acting opioid agonist codeine. CONCLUSION: Pharmacological tests have revealed a new pharmacodynamic effect of the Erigeron complex, namely an antitussive effect. Its activity was most pronounced in comparison with all previously tested compounds from other medicinal plants and approached the effect of codeine, the most potent antitussive used in clinical practice. The results provide the scientific basis for the application of this herb in traditional medicine.
Assuntos
Erigeron/química , Polifenóis/farmacologia , Polissacarídeos/farmacologia , Proteínas/farmacologia , Animais , Antitussígenos/química , Antitussígenos/isolamento & purificação , Antitussígenos/farmacologia , Codeína/farmacologia , Tosse/tratamento farmacológico , Relação Dose-Resposta a Droga , Cobaias , Masculino , Polifenóis/química , Polifenóis/isolamento & purificação , Polissacarídeos/administração & dosagem , Polissacarídeos/química , Polissacarídeos/isolamento & purificação , Proteínas/química , Proteínas/isolamento & purificaçãoRESUMO
BACKGROUND PEGylation, defined as the covalent attachment of polyethylene glycol, allows the synthesis of PEGylated therapeutic proteins with enhanced physicochemical properties. Traditional alkylating Nterminal PEGylation reactions on amine groups involve the use of modified linear mono-methoxy polyethylene glycol (mPEG) molecules looking for the synthesis of mono-PEGylated products. However, this approach requires different purification steps since inevitably undesired cross-linked products are synthesized. Herein, we propose the use of reactive aqueous two-phase systems (ATPS) to produce and purify PEGylated therapeutic conjugates using Ribonuclease A (RNase A) as a model protein. RESULTS: Selected linear 5 kDa and 20 kDa mPEG potassium phosphate systems were produced according to equilibrium data obtained from constructed binodal curves. All reactive systems were able to generate biphasic systems and to PEGylate RNase A. Two 5 kDa and two 20 kDa systems were selected based on the reaction yield percentage and the feasibility of purifying the mono-PEGylated RNase A from the diPEGylated and native RNase A by contrasting the differences in their partition behaviors. The remnant biological activity was of 94% and of 100% for the mono-PEGylated RNase A purified from the 5 kDa and 20 kDa mPEG systems when compared to the mono-PEGylated conjugate obtained by standard procurement methods.
Assuntos
Polietilenoglicóis/química , Proteínas/isolamento & purificação , Proteínas/químicaRESUMO
Supercritical fluid chromatography (SFC) has unique separative characteristics distinguished from those of HPLC and gas chromatography. At present, SFC is widely used and there are many applications in various biological, medical, and pharmaceutical fields. In this review, we focus on recently developed novel techniques related to SFC separation including: new column stationary phases, microfluidics, two-dimensional separation, and gas-liquid separation. In addition, we discuss the application of SFC using a water-containing modifier to biological molecules such as amino acids, peptides, and small proteins that had been challenging analytes.
Assuntos
Aminoácidos/isolamento & purificação , Peptídeos/isolamento & purificação , Proteínas/isolamento & purificação , Aminoácidos/química , Cromatografia com Fluido Supercrítico , Peptídeos/química , Proteínas/químicaRESUMO
Cysteine S-nitrosation mediates NO signaling and protein function under pathophysiological conditions. Herein, we provide a detailed protocol regarding the organic mercury chemoselective enrichment of S-nitrosated proteins and peptides. We discuss key aspects of the enrichment strategy and provide technical tips for the best performance of the experimental protocol.
Assuntos
Mercúrio/química , Nitratos , Proteínas , Proteômica/métodos , Cromatografia , Cisteína/análise , Cisteína/isolamento & purificação , Cisteína/metabolismo , Nitratos/análise , Nitratos/isolamento & purificação , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Nitrosação , Peptídeos/análise , Peptídeos/isolamento & purificação , Peptídeos/metabolismo , Proteínas/análise , Proteínas/isolamento & purificação , Proteínas/metabolismoRESUMO
In chronic lymphocytic leukemia (CLL) and very likely all cancer types, extracellular vesicles (EVs) are a common mechanism by which intercellular messages are communicated between normal, diseased, and transformed cells. Studies of EVs in CLL and other cancers have great variability and often lack reproducibility. For CLL patient plasma and cell lines, we sought to characterize current approaches used in isolating EV products and understand whether cell culture-conditioned media or complex biological fluids confound results. Utilizing nanoparticle tracking analysis, protein quantification, and electron microscopy, we show that ultracentrifugation with an OptiPrep cushion can effectively minimize contaminants from starting materials including plasma and conditioned media of CLL cell lines grown in EV-depleted complete RPMI media but not grown in the serum-free media AIM V commonly used in CLL experimental work. Moreover, we confirm the benefit of including 25 mM trehalose in PBS during EV isolation steps to reduce EV aggregation, to preserve function for downstream applications and characterization. Furthermore, we report the highest particles/µg EVs were obtained from our CLL cell lines utilizing the CELLine bioreactor flask. Finally, we optimized a proliferation assay that offers a functional evaluation of our EVs with minimal sample requirements.
Assuntos
Técnicas de Química Analítica/métodos , Vesículas Extracelulares , Proteínas/isolamento & purificação , Linhagem Celular , Meios de Cultivo Condicionados , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/patologia , Humanos , Leucemia Linfocítica Crônica de Células B , Microscopia Eletrônica/métodos , Nanopartículas , Imagem Individual de Molécula/instrumentação , Imagem Individual de Molécula/métodosRESUMO
This study investigates the antioxidant activities of lipid, protein, and carbohydrate extracts from the marine mollusk Perna canaliculus. Lipids were extracted using acetone, which was followed by protein extraction using the broad-spectrum enzyme Alcalase and then carbohydrate extraction using cetylpyridinium chloride. Eighty white BALB/c mice were divided into eight groups according to the administered extracts. Groups 1 and 5 were the control and toxin control groups, respectively. Groups 2, 3, and 4 were administered lipid, protein, and carbohydrate extracts, respectively. The other groups were administered P. canaliculus extracts as well as gentamicin and acetaminophen, known as ethanolic extracts, derived from Nerium oleander to induce oxidation stress. All groups showed significant improvements in body weight (p < 0.05). The lipid extract group showed a significant decrease in low-density lipoprotein cholesterol (p < 0.05) and a significant increase in high-density lipoprotein cholesterol (p < 0.05). After the toxin injection, all groups treated with P. canaliculus extracts showed increased antioxidant effects on hepatocytes (p < 0.05). The lipid extracts induced antioxidant effects to protect the kidney by increasing lipid peroxidation (p < 0.05) and catalase activities (p < 0.05). Also, protein extracts showed antioxidant effects by increasing glutathione and catalase levels significantly (p < 0.005). In conclusion, P. canaliculus extracts, especially lipids and proteins, have potent antioxidant activities that protect vital organs from oxidation stress.
Assuntos
Antioxidantes/administração & dosagem , Carboidratos/administração & dosagem , Lipídeos/administração & dosagem , Perna (Organismo)/química , Proteínas/administração & dosagem , Animais , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Produtos Biológicos/isolamento & purificação , Carboidratos/isolamento & purificação , Carboidratos/farmacologia , Catalase/metabolismo , Etanol/administração & dosagem , Etanol/farmacologia , Glutationa/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/isolamento & purificação , Lipídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Nerium/química , Estresse Oxidativo/efeitos dos fármacos , Proteínas/isolamento & purificação , Proteínas/farmacologiaRESUMO
This study aims to investigate effects of ultrasound assisted extraction on the abalone viscera protein extraction rate and iron-chelating activity of peptides. The optimal conditions for ultrasound assisted extraction by response surface methodology was at sodium hydroxide concentration 14 g/kg, ultrasonic power 428 W and extraction time 52 min. Under the optimal conditions, protein extraction rate was 64.89%, compared with alkaline extraction of 55.67%. The iron-chelating activity of peptides affected by ultrasound technology was further evaluated by iron-chelating rate, FTIR spectroscopy and LC-HRMS/MS. Alcalase was the suitable enzyme for the preparation of iron-chelating peptides from two abalone viscera proteins, showing no significant difference between their iron-chelating rate of 16.24% (ultrasound assisted extraction) and 16.60% (alkaline extraction). Iron binding sites from the two hydrolysates include amino and carboxylate terminal groups and peptide bond of the peptide backbone as well as amino, imine and carboxylate from side chain groups. Moreover, 24 iron-chelating peptides were identified from hydrolysate (alcalase, ultrasound assisted extraction), which were different from the 27 iron-chelating peptides from hydrolysate (alcalase, alkaline extraction). This study suggests the application of ultrasound technology in the generation of abalone viscera-derived iron-chelating peptides which have the ability to combat iron deficiency.
Assuntos
Fracionamento Químico/métodos , Quelantes de Ferro/isolamento & purificação , Quelantes de Ferro/farmacologia , Moluscos , Proteínas/isolamento & purificação , Proteínas/farmacologia , Ondas Ultrassônicas , Vísceras , AnimaisRESUMO
Reliable separation of peptides, amino acids and proteins as accurate as possible with the maximum conformation and biological activity is crucial and essential for drug discovery. Polysaccharide, as one of the most abundant natural biopolymers with optical activity on earth, is easy to be functionalized due to lots of hydroxyl groups on glucose units. Over the last few decades, polysaccharide derivatives are gradually employed as effective separation media. The highly-ordered helical structure contributes to complex, diverse molecular recognition ability, allowing polysaccharide derivatives to selectively interact with different analytes. This article reviews the development, application and prospects of polysaccharides as separation media in the separation of proteins, peptides and amino acids in recent years. The chiral molecules mechanism, advantages, limitations, development status and challenges faced by polysaccharides as separation media in molecular recognition are summarized. Meanwhile, the direction of its continued development and future prospects are also discussed.
Assuntos
Aminoácidos/isolamento & purificação , Peptídeos/isolamento & purificação , Polissacarídeos/química , Proteínas/isolamento & purificação , Aminoácidos/química , Configuração de Carboidratos , Fracionamento Químico , Peptídeos/química , Conformação Proteica , Proteínas/química , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Polymer-based monolithic high-performance liquid chromatography (HPLC) columns are normally obtained by conventional free-radical polymerization. Despite being straightforward, this approach has serious limitations with respect to controlling the structural homogeneity of the monolith. Herein, we explore a reversible addition-fragmentation chain transfer (RAFT) polymerization method for the fabrication of porous polymers with well-defined porous morphology and surface chemistry in a confined 200 µm internal diameter (ID) capillary format. This is achieved via the controlled polymerization-induced phase separation (controlled PIPS) synthesis of poly(styrene-co-divinylbenzene) in the presence of a RAFT agent dissolved in an organic solvent. The effects of the radical initiator/RAFT molar ratio as well as the nature and amount of the organic solvent were studied to target cross-linked porous polymers that were chemically bonded to the inner wall of a modified silica-fused capillary. The morphological and surface properties of the obtained polymers were thoroughly characterized by in situ nuclear magnetic resonance (NMR) experiments, nitrogen adsorption-desorption experiments, elemental analyses, field-emission scanning electron microscopy (FESEM), scanning electron microscopy-energy-dispersive X-ray (SEM-EDX) spectroscopy, and X-ray photoelectron spectroscopy (XPS) as well as time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealing the physicochemical properties of these styrene-based materials. When compared with conventional synthetic methods, the controlled-PIPS approach affects the kinetics of polymerization by delaying the onset of phase separation, enabling the construction of materials with a smaller pore size. The results demonstrated the potential of the controlled-PIPS approach for the design of porous monolithic columns suitable for liquid separation of biomolecules such as peptides and proteins.
Assuntos
Cromatografia Líquida/métodos , Polimerização , Peptídeos/isolamento & purificação , Porosidade , Proteínas/isolamento & purificação , Solventes/químicaRESUMO
A genome-inspired route to aptamer discovery that expands the sequence space beyond that available in traditional, combinatorial selection approaches is investigated for discovery of DNA-protein interactions in cancer. These interactions could then serve as the basis for new DNA aptamers to cancer-related proteins. The genome-inspired approach uses specific DNA sequences from the human genome to capture proteins from biological protein pools. The use of naturally occurring DNA sequences takes advantage of biological evolution of DNA sequences that bind to specific proteins to perform biological functions. Linking aptamer discovery to nature increa`ses the chances of uncovering protein-DNA affinity binding interactions that have biological significance as well as analytical utility. Here, the focus is on genomic, G-rich sequences that can form G-quadruplex (G4) structures. These structures are underrepresented in combinatorial libraries used for conventional aptamer selection. Additionally, G4-forming sequences are prone to inefficient PCR amplification, further biasing aptamer selection away from these structures. Nature provides a large diversity of G4-forming sequences throughout the human genome. They are prevalent in gene promoter regions, especially in oncogene promoters, and are therefore promising candidates for aptamers to regulatory proteins in cancer. The present work investigates protein capture from nuclear and cytoplasmic extracts of the breast cancer cell line MDA-MB-468 by G4-forming sequences from the CMYC, RB, and VEGF gene promoters. The studies included the effects of modifications of the VEGF sequence on the selectivity of protein capture, from which we identified promising aptamer candidates, subject to further refinement, to the proteins nucleolin and RPL19, both of which play important regulatory functions related to cancer.
Assuntos
Aptâmeros de Nucleotídeos/genética , Quadruplex G , Proteínas/isolamento & purificação , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Regiões Promotoras Genéticas , Proteínas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Major histocompatibility complexes (MHC) play a critical role in immunity by presenting peptides on the cell surface for T cell recognition. Identification of these peptides can be valuable to develop vaccines or immunotherapeutic strategies for infectious diseases and cancers. Mass spectrometry is the only tool available for unbiased identification of the immunopeptidome. Here, we describe a protocol for purification and identification of MHC class I peptides, including in-house purification of anti-MHC-antibody from hybridoma cells and the LC-MS/MS analysis of MHC-I bound peptides.
Assuntos
Antígenos de Histocompatibilidade Classe I/isolamento & purificação , Peptídeos/imunologia , Peptídeos/isolamento & purificação , Linhagem Celular , Membrana Celular/imunologia , Cromatografia Líquida/métodos , Antígenos HLA/imunologia , Antígenos HLA/isolamento & purificação , Antígenos de Histocompatibilidade Classe I/química , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Complexo Principal de Histocompatibilidade/imunologia , Peptídeos/química , Proteínas/isolamento & purificação , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Biomolecular condensates formed by liquid-liquid phase separation (LLPS) are considered one of the early compartmentalization strategies of cells, which still prevail today forming nonmembranous compartments in biological cells. Studies of the effect of high pressures, such as those encountered in the subsurface salt lakes of Mars or in the depths of the subseafloor on Earth, on biomolecular LLPS will contribute to questions of protocell formation under prebiotic conditions. We investigated the effects of extreme environmental conditions, focusing on highly aggressive Martian salts (perchlorate and sulfate) and high pressure, on the formation of biomolecular condensates of proteins. Our data show that the driving force for phase separation of proteins is not only sensitively dictated by their amino acid sequence but also strongly influenced by the type of salt and its concentration. At high salinity, as encountered in Martian soil and similar harsh environments on Earth, attractive short-range interactions, ion correlation effects, hydrophobic, and π-driven interactions can sustain LLPS for suitable polypeptide sequences. Our results also show that salts across the Hofmeister series have a differential effect on shifting the boundary of immiscibility that determines phase separation. In addition, we show that confinement mimicking cracks in sediments and subsurface saline water pools in the Antarctica or on Mars can dramatically stabilize liquid phase droplets, leading to an increase in the temperature and pressure stability of the droplet phase.
Assuntos
Meio Ambiente Extraterreno/química , Sais/química , Sequência de Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Extração Líquido-Líquido , Marte , Proteínas/química , Proteínas/isolamento & purificação , Salinidade , TemperaturaRESUMO
In the present protocol, extracellular vesicles (EVs) released from a primary culture of human umbilical cord mesenchymal stem cells (MSCs) were isolated by ultracentrifugation processes, characterized by transmission electron microscopy (TEM) and measured by nanoparticle tracking analysis (NTA). Protein was extracted from EVs using RIPA buffer and then was assessed for integrity. The proteomic content of the total EV protein samples was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after labeling by tandem mass tag (TMT). This combined approach allowed the development of an effective strategy to study the protein cargo from MSC-derived EVs.