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Milk is a rich source of biologically important proteins and peptides. In addition, milk contains a variety of extracellular vesicles (EVs), including exosomes, that carry their own proteome cargo. EVs are essential for cell-cell communication and modulation of biological processes. They act as nature carriers of bioactive proteins/peptides in targeted delivery during various physiological and pathological conditions. Identification of the proteins and protein-derived peptides in milk and EVs and recognition of their biological activities and functions had a tremendous impact on food industry, medicine research, and clinical applications. Advanced separation methods, mass spectrometry (MS)-based proteomic approaches and innovative biostatistical procedures allowed for characterization of milk protein isoforms, genetic/splice variants, posttranslational modifications and their key roles, and contributed to novel discoveries. This review article discusses recently published developments in separation and identification of bioactive proteins/peptides from milk and milk EVs, including MS-based proteomic approaches.
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Vesículas Extracelulares , Proteínas de la Leche , Animales , Proteínas de la Leche/análisis , Proteómica/métodos , Leche/química , Vesículas Extracelulares/química , Péptidos/análisisRESUMEN
Probiotics can affect human health, keep the balance between beneficial and pathogenic bacteria, and their colonizing abilities enable the enhancement of the epithelial barrier, preventing the invasion of pathogens. Health benefits of probiotics were related to allergy, depression, eczema, cancer, obesity, inflammatory diseases, viral infections, and immune regulation. Probiotic bacterial cells contain various proteins that function as effector molecules, and explaining their roles in probiotic actions is a key to developing efficient and targeted treatments for various disorders. Systematic proteomic studies of probiotic proteins (probioproteomics) can provide information about the type of proteins involved, their expression levels, and the pathological changes. Advanced proteomic methods with mass spectrometry instrumentation and bioinformatics can point out potential candidates of next-generation probiotics that are regulated under pharmaceutical frameworks. In addition, the application of proteomics with other omics methods creates a powerful tool that can expand our understanding about diverse probiotic functionality. In this review, proteomic strategies for identification/quantitation of the proteins in probiotic bacteria were overviewed. The types of probiotic proteins investigated by proteomics were described, such as intracellular proteins, surface proteins, secreted proteins, and the proteins of extracellular vesicles. Examples of pathological conditions in which probiotic bacteria played crucial roles were discussed.
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Proteínas Bacterianas , Probióticos , Proteómica , Proteómica/métodos , Humanos , Proteínas Bacterianas/metabolismo , Bacterias/metabolismo , AnimalesRESUMEN
Immunoglobulins in bovine colostrum were separated and fractionated from other proteins using the method and instrumentation developed in our laboratory. The proposed separation was based on bidirectional isotachophoresis/moving boundary electrophoresis with electrofocusing of the analytes in a pH gradient from 3.9 to 10.1. The preparative instrumentation included the trapezoidal non-woven fabric that served as separation space with divergent continuous flow. The defatted and casein precipitate-free colostrum supernatant was loaded directly into the instrument without any additional colostrum pre-preparation. Immunoglobulin G was fractionated from other immune proteins such as bovine serum albumin, ß-lactoglobulin, and α-lactalbumin, and was continuously collected in separated fractions over 3 h. The fractions were further processed, and isolated immunoglobulin G in the liquid fractions was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by re-focusing in gel isoelectric focusing. Separated immunoglobulin G was detected in seven fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a gradually decreased concentration in the fractions. Re-focusing of the proteins in the fractions by gel isoelectric focusing revealed multiple separated zones of immunoglobulin G with the isoelectric point values covering the range from 5.4 to 7.2. Each fraction contained distinct zones with gradually increased isoelectric point values and decreased concentrations from fraction to fraction.
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Caseínas , Calostro , Femenino , Embarazo , Humanos , Calostro/química , Dodecil Sulfato de Sodio , Focalización Isoeléctrica/métodos , Caseínas/análisis , Electroforesis en Gel de Poliacrilamida , Inmunoglobulina G , InmunoglobulinasRESUMEN
The focus of this review is on the proteomic approaches applied to the study of the qualitative/quantitative changes in mitochondrial proteins that are related to impaired mitochondrial function and consequently different types of pathologies. Proteomic techniques developed in recent years have created a powerful tool for the characterization of both static and dynamic proteomes. They can detect protein-protein interactions and a broad repertoire of post-translation modifications that play pivotal roles in mitochondrial regulation, maintenance and proper function. Based on accumulated proteomic data, conclusions can be derived on how to proceed in disease prevention and treatment. In addition, this article will present an overview of the recently published proteomic papers that deal with the regulatory roles of post-translational modifications of mitochondrial proteins and specifically with cardiovascular diseases connected to mitochondrial dysfunction.
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Enfermedades Cardiovasculares , Proteómica , Humanos , Proteómica/métodos , Proteoma/metabolismo , Enfermedades Cardiovasculares/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Procesamiento Proteico-PostraduccionalRESUMEN
We constructed a preparative instrumentation and developed the methods that are based on separation of the samples by bidirectional isotachophoresis/moving boundary electrophoresis in continuous divergent flow. The described instrumentation can be used for a variety of the samples, however, it can be easily optimized and tailored for the specific sample. The trapezoid separation bed from nonwoven textile exhibited minimum adsorption effect for sample and it can be used repeatedly. By the addition of different spacers via separation space inlets, the sections of pH gradient can be modified to enhance the separation. The liquid flow from two inlets positioned on each side of the sample inlet prevented the contact of the sample with anolyte and catholyte at the analysis beginning. One pair of thin electrodes (graphite and stainless-steel) was placed at the separation space output. The electrode products were washed out into drains without disturbing the focusing process. The influence of EOF was managed by tilting the separation bed in the direction from cathodic to anodic side. The components of spirulina supernatant and color pI markers were separated in the pH gradient from 3.9 to 10.1. pH gradient was stable for at least 4.5 h and spirulina supernatant from about 0.12 g of dry powder was processed. Compared to other preparative methods used for spirulina separation, the presented method/instrumentation working with a continuous divergent flow had essential advantages. The efficient separation was fast, and no intermediate steps were necessary to obtain liquid fractions with separated components compatible with further biological experiments.
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Isotacoforesis , ElectrodosRESUMEN
Cardiovascular disease is the main cause of death worldwide, making it crucial to search for new therapies to mitigate major adverse cardiac events (MACEs) after a cardiac ischemic episode. Drugs in the class of the glucagon-like peptide-1 receptor agonists (GLP1Ra) have demonstrated benefits for heart function and reduced the incidence of MACE in patients with diabetes. Previously, we demonstrated that a short-acting GLP1Ra known as DMB (2-quinoxalinamine, 6,7-dichloro-N-[1,1-dimethylethyl]-3-[methylsulfonyl]-,6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline or compound 2, Sigma) also mitigates adverse postinfarction left ventricular remodeling and cardiac dysfunction in lean mice through activation of parkin-mediated mitophagy following infarction. Here, we combined proteomics with in silico analysis to characterize the range of effects of DMB in vivo throughout the course of early postinfarction remodeling. We demonstrate that the mitochondrion is a key target of DMB and mitochondrial respiration, oxidative phosphorylation and metabolic processes such as glycolysis and fatty acid beta-oxidation are the main biological processes being regulated by this compound in the heart. Moreover, the overexpression of proteins with hub properties identified by protein-protein interaction networks, such as Atp2a2, may also be important to the mechanism of action of DMB. Data are available via ProteomeXchange with identifier PXD027867.
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Ventrículos Cardíacos/metabolismo , Mitocondrias Cardíacas/metabolismo , Proteómica/métodos , Quinoxalinas/administración & dosificación , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Remodelación Ventricular/efectos de los fármacos , Animales , Biología Computacional , Modelos Animales de Enfermedad , Receptor del Péptido 1 Similar al Glucagón/agonistas , Glucólisis , Masculino , Ratones , Fosforilación Oxidativa , Mapas de Interacción de Proteínas , Quinoxalinas/farmacologíaRESUMEN
The effects of ER stress on protein secretion by cardiac myocytes are not well understood. In this study, the ER stressor thapsigargin (TG), which depletes ER calcium, induced death of cultured neonatal rat ventricular myocytes (NRVMs) in high media volume but fostered protection in low media volume. In contrast, another ER stressor, tunicamycin (TM), a protein glycosylation inhibitor, induced NRVM death in all media volumes, suggesting that protective proteins were secreted in response to TG but not TM. Proteomic analyses of TG- and TM-conditioned media showed that the secretion of most proteins was inhibited by TG and TM; however, secretion of several ER-resident proteins, including GRP78 was increased by TG but not TM. Simulated ischemia, which decreases ER/SR calcium also increased secretion of these proteins. Mechanistically, secreted GRP78 was shown to enhance survival of NRVMs by collaborating with a cell-surface protein, CRIPTO, to activate protective AKT signaling and to inhibit death-promoting SMAD2 signaling. Thus, proteins secreted during ER stress mediated by ER calcium depletion can enhance cardiac myocyte viability.
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Estrés del Retículo Endoplásmico , Miocitos Cardíacos/metabolismo , Proteoma , Proteómica , Animales , Apoptosis , Comunicación Autocrina , Biomarcadores , Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Supervivencia Celular , Células Cultivadas , Susceptibilidad a Enfermedades , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico/efectos de los fármacos , Factor de Crecimiento Epidérmico/metabolismo , Glicoproteínas de Membrana/metabolismo , Ratones , Miocitos Cardíacos/efectos de los fármacos , Proteínas de Neoplasias/metabolismo , Comunicación Paracrina , Proteómica/métodos , Ratas , Retículo Sarcoplasmático/metabolismo , Transducción de Señal/efectos de los fármacos , Tapsigargina/farmacologíaRESUMEN
Continuous flow electrophoretic separation with continuous sample loading provides the advantage of processing volumes of any sizes, as well as the benefit of a real-time monitoring and optimization of the separation process. In addition, the spatial separation of the sample enables collecting multiple separated components simultaneously and in a continuous manner. The separation is usually performed in mild buffers without organic solvents and detergents (sample biological activity is retained) and it is carried out without usage of a solid support in the separation space preventing the interaction of the sample with it (high sample recovery). The method is used for the separation of proteins/peptides in proteomic applications, and its great applicability is to the separation of the cells, cellular organelles, vesicles, membrane fragments, and DNA. This review focuses on the electrophoretic separation performed in a continuous flow and it describes various electrophoretic modes and instrumental setups. Recent developments in methodology and instrumentation, the integration with other techniques, and the application to the biological sample analysis are discussed as well.
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Electroforesis , ADN/aislamiento & purificación , Electroforesis/instrumentación , Electroforesis/métodos , Diseño de Equipo , Péptidos/aislamiento & purificación , Proteínas/aislamiento & purificaciónRESUMEN
INTRODUCTION: Protein synthesis is the outcome of tightly regulated gene expression which is responsive to a variety of conditions. Efforts are ongoing to monitor individual stages of protein synthesis to ensure maximum efficiency and accuracy. Due to post-transcriptional regulation mechanisms, the correlation between translatome and proteome is higher than between transcriptome and proteome. However, the most accurate approach to assess the key modulators and final protein expression is directly by using proteomics. Areas covered: This review covers various proteomic strategies that were used to better understand post-transcriptional regulation, specifically during and early after translation. The methods that identify both regulatory proteins associated with translational components and newly synthesized proteins are discussed. Expert commentary: Emerging proteomic approaches make it possible to monitor protein dynamics in cells, tissues and whole animals. The ability to detect alteration in protein abundance soon after their synthesis enables earlier recognition of disease causing factors and candidates to prevent/rectify disease phenotype.
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Biosíntesis de Proteínas/genética , Proteómica , Ribosomas/genética , Animales , Investigación Biomédica/métodos , Regulación de la Expresión Génica/genéticaRESUMEN
The alteration in proteome composition induced by environmental changes and various pathologies is accompanied by the modifications of proteins by specific cotranslational and PTMs. The type and site stoichiometry of PTMs can affect protein functions, alter cell signaling, and can have acute and chronic effects. The particular interest is drawn to those amino acid residues that can undergo several different PTMs. We hypothesize that these selected amino acid residues are biologically rare and act within the cell as molecular switches. There are, at least, 12 various lysine modifications currently known, several of them have been shown to be competitive and they influence the ability of a particular lysine to be modified by a different PTM. In this review, we discuss the PTMs that occur on lysine, specifically neddylation and sumoylation, and the proteomic approaches that can be applied for the identification and quantification of these PTMs. Of interest are the emerging roles for these modifications in heart disease and what can be inferred from work in other cell types and organs.
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Cardiopatías/metabolismo , Lisina , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Secuencia de Aminoácidos , Animales , Bovinos , Células Cultivadas , Humanos , Lisina/análisis , Lisina/química , Lisina/metabolismo , Ratones , Datos de Secuencia Molecular , Miocardio/patología , Miocitos Cardíacos/química , Miocitos Cardíacos/metabolismo , RatasRESUMEN
This study concentrates on development of instrumentation for focusing and separation of analytes in continuous flow. It is based on bidirectional ITP working in wide pH range with separation space of closed void channel of trapezoidal shape and continuous supply of sample. The novel instrumentation is working with electrolyte system formulated previously and on the contrary to devices currently available, it allows preparative separation and concentration of cationic, anionic, and amphoteric analytes simultaneously and in wide pH range. The formation of sharp edges at zone boundaries as well as low conductivity zones are avoided in suggested system and thus, local overheating is eliminated allowing for high current densities at initial stages of focusing. This results in high focusing speed and reduction of analysis time, which is particularly advantageous for separations performed in continuous flow systems. The closed void channel is designed to avoid basic obstacles related to liquid leakage, bubbles formation, contacts with electrodes, channel height and complicated assembling. The performance of designed instrumentation and focusing dynamics were tested by using colored low molecular mass pH indicators for local pH determination, focusing pattern, and completion. In addition, feasibility and separation efficiency were demonstrated by focusing of cytochrome C and myoglobin. The collection of fractions at instrument output allows for subsequent analysis and identification of sample components that are concentrated and conveniently in form of solution for further processing. Since the instrumentation operates with commercially available simple defined buffers and compounds without need of carrier ampholytes background, it is economically favorable.
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Isotacoforesis/instrumentación , Isotacoforesis/métodos , Colorantes/química , Colorantes/aislamiento & purificación , Diseño de Equipo , Estudios de Factibilidad , Concentración de Iones de Hidrógeno , Modelos Químicos , Mioglobina/química , Mioglobina/aislamiento & purificaciónRESUMEN
In this paper, we suggest new electrolyte system for fast preparative electrofocusing in wide pH range. It is based on bidirectional ITP with multiple counterions and spacers created by commercially available defined simple buffers. The migration course of proposed focusing model can be simulated in advance by using separation conditions and electrolyte components that are consequently applied during the experiments. The suggested electrolyte system allows high current densities at the initial stages of focusing without danger of local overheating, which strongly reduces the time needed for analysis completion. The performance of the electrolyte system is demonstrated by the focusing of synthetic colored low molecular weight indicators and proteins in the arrangements with both linear narrow strip and nonwoven fabric sheet with continuous flow.
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Electrólitos/química , Focalización Isoeléctrica/métodos , Isotacoforesis/métodos , Colorantes/química , Colorantes/aislamiento & purificación , Concentración de Iones de HidrógenoRESUMEN
Over 400 different types of post-translational modifications (PTMs) have been reported and over 200 various types of PTMs have been discovered using mass spectrometry (MS)-based proteomics. MS-based proteomics has proven to be a powerful method capable of global PTM mapping with the identification of modified proteins/peptides, the localization of PTM sites and PTM quantitation. PTMs play regulatory roles in protein functions, activities and interactions in various heart related diseases, such as ischemia/reperfusion injury, cardiomyopathy and heart failure. The recognition of PTMs that are specific to cardiovascular pathology and the clarification of the mechanisms underlying these PTMs at molecular levels are crucial for discovery of novel biomarkers and application in a clinical setting. With sensitive MS instrumentation and novel biostatistical methods for precise processing of the data, low-abundance PTMs can be successfully detected and the beneficial or unfavorable effects of specific PTMs on cardiac function can be determined. Moreover, computational proteomic strategies that can predict PTM sites based on MS data have gained an increasing interest and can contribute to characterization of PTM profiles in cardiovascular disorders. More recently, machine learning- and deep learning-based methods have been employed to predict the locations of PTMs and explore PTM crosstalk. In this review article, the types of PTMs are briefly overviewed, approaches for PTM identification/quantitation in MS-based proteomics are discussed and recently published proteomic studies on PTMs associated with cardiovascular diseases are included.
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The proteins secreted by various cells (the secretomes) are a potential rich source of biomarkers as they reflect various states of the cells at real time and at given conditions. To have accessible, sufficient and reliable protein markers is desirable as they mark various stages of disease development and their presence/absence can be used for diagnosis, prognosis, risk stratification and therapeutic monitoring. As direct analysis of blood/plasma, a common and noninvasive patient screening method, can be difficult for candidate protein biomarker identification, the alternative/complementary approaches are required, one of them is the analysis of secretomes in cell conditioned media in vitro. As the proteins secreted by cells as a response to various stimuli are most likely secreted into blood/plasma, the identification and pre-selection of candidate protein biomarkers from cell secretomes with subsequent validation of their presence at higher levels in serum/plasma is a promising approach. In this review, we discuss the proteins secreted by three progenitor cell types (smooth muscle, endothelial and cardiac progenitor cells) and two adult cell types (neonatal rat ventrical myocytes and smooth muscle cells) which can be relevant to cardiovascular research and which have been recently published in the literature. We found, at least for secretome studies included in this review, that secretomes of progenitor and adult cells overlap by 48% but the secretomes are very distinct among progenitor cell themselves as well as between adult cells. In addition, we compared secreted proteins to protein identifications listed in the Human Plasma PeptideAtlas and in two reports with cardiovascular-related proteins and we performed the extensive literature search to find if any of these secreted proteins were identified in a biomarker study. As expected, many proteins have been identified as biomarkers in cancer but 18 proteins (out of 62) have been tested as biomarkers in cardiovascular diseases as well.
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Proteínas de Neoplasias/sangre , Proteínas/metabolismo , Proteómica/métodos , Animales , Biomarcadores de Tumor/sangre , Biomarcadores de Tumor/metabolismo , Proteínas Sanguíneas , Células Endoteliales/metabolismo , Humanos , Músculo Liso/metabolismo , Miocitos Cardíacos/metabolismo , Ratas , Células Madre/metabolismoRESUMEN
Protein isoforms/splice variants can play important roles in various biological processes and can potentially be used as biomarkers or therapeutic targets/mediators. Thus, there is a need for efficient and, importantly, accurate methods to distinguish and quantify specific protein isoforms. Since protein isoforms can share a high percentage of amino acid sequence homology and dramatically differ in their cellular concentration, the task for accuracy and efficiency in methodology and instrumentation is challenging. The analysis of intact proteins has been perceived to provide a more accurate and complete result for isoform identification/quantification in comparison to analysis of the corresponding peptides that arise from protein enzymatic digestion. Recently, novel approaches have been explored and developed that can possess the accuracy and reliability important for protein isoform differentiation and isoform-specific peptide targeting. In this review, we discuss the recent development in methodology and instrumentation for enhanced detection of protein isoforms as well as the examples of their biological importance.
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Cromatografía Liquida/métodos , Espectrometría de Masas/métodos , Isoformas de Proteínas/análisis , Proteómica/métodos , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Isoformas de Proteínas/química , Alineación de SecuenciaRESUMEN
BACKGROUND: The aqueous humor (AH), a liquid of the anterior and posterior chamber of the eye, comprises many proteins with various roles and important biological functions. Many of these proteins have not been identified yet and their functions in AH are still unknown. Recently, our laboratory published the protein database of AH obtained from healthy rabbits which expanded known protein identifications by 65%. Our present study extends our previous work and analyses AH following two types of cataract surgery incision procedures (clear corneal and limbal incisions) by using two dimensional gel electrophoresis (2-DE) and liquid chromatography tandem mass spectrometry (LC-MS/MS). Although both incision protocols are commonly used during cataract surgeries, the difference in protein composition and their release into AH following each surgery has never been systematically compared and remains unclear. The first step, which is the focus of this work, is to assess the scale of the protein change, at which time does maximum release occurs and when possible, to identify protein changes. RESULTS: Samples of AH obtained prior to surgery and at different time points (0.5, 2, 12, 24 and 48 hours) following surgery (n = 3/protocol) underwent protein concentration determination, 2-DE and LC-MS/MS. There was a large (9.7 to 31.2 mg/mL) and rapid (~0.5 hour) influx of proteins into AH following either incision with a return to baseline quantities after 12 hours and 24 hours for clear corneal and limbal incision, respectively. We identified 80 non-redundant proteins, and compared to our previous study on healthy AH, 67.5% of proteins were found to be surgery-specific. In addition, 51% of those proteins have been found either in clear corneal (20%) or limbal incision (31%) samples. CONCLUSIONS: Our results imply that a mechanism of protein release into AH after surgery is a global response to the surgery rather than increase in amount of protective proteins found in healthy AH and a mechanism of protein release for each type of incision procedure could be different. Although the total protein concentration was increased (at 0.5 and 2 hour time points and between types of surgery) many of 2-DE protein spots were similar based on 2-DE and MS analyses, and only a small number of protein spots changed with either the time points or surgical conditions (0.4 -1.9%). This suggests that the high protein content is due to an increase in the concentration of the same proteins with only a few unique proteins being altered per time point and with the different surgery type. This is the first report on the comparison of AH protein composition following two different cataract surgery procedures and it establishes the basis for better understanding of protein release into AH during events such as cataract surgery or other possible intervention to the eyes.
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In the heart, the proteomes secreted by both cardiac stem cells (CSCs) and cardiac myocytes could act synergistically, but the identification and functionality of the proteins comprising the individual secretomes have not yet been described. In this study, we have identified proteins present in the media obtained from cultured rat CSCs and from cultured neonatal rat ventricular myocytes (NRVMs) and compared them with proteins identified in the media alone. Briefly, 83 unique proteins were identified after analysis by RPLC and MS. In total 49 and 23% were NRVM-specific or CSC-specific proteins, respectively, and 63% of total 83 proteins were integral plasma membrane and/or known secreted proteins. Fifteen proteins met our criteria for paracrine/autocrine factors: (i) robust protein identification, (ii) cell specific and (iii) known to be secreted. Most of these proteins have not been previously linked to stem cells. NRVM-specific proteins atrial natriuretic factor (ANP) and connective tissue growth factor, and CSC-specific protein interleukin-1 receptor-like 1 (ST2) were found to affect rat CSC proliferation. These findings suggest that relative concentration of each protein may be crucial for cellular intertalk and for the final outcome of cardiac cell therapy.
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Miocitos Cardíacos/química , Proteoma/análisis , Células Madre/química , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Proliferación Celular , Células Cultivadas , Datos de Secuencia Molecular , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteoma/química , Proteoma/metabolismo , Ratas , Ratas Endogámicas WKY , Células Madre/metabolismoRESUMEN
Efficient separation method is a crucial part of the process in which components of highly complex biological sample are identified and characterized. Based on the principles of recently newly established electrophoretic method called divergent flow IEF (DF IEF), we have tested the DF IEF instrument which is able to operate without the use of background carrier ampholytes. We have verified that during separation and focusing of sample consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be created by autofocusing of the sample components themselves without any addition of carrier ampholytes. In DF IEF, the proteins are separated, desalted and concentrated in one step. The fractions of yeast lysate sample, collected at the DF IEF output and subjected to gel IEF, contained the zones of proteins gradually covering the pI values from 3.7 to 8.5. In our experimental arrangement, the highest number of proteins has been found in fractions with pI values around 5.3 as detected by polyacrylamide gel IEF with CBB staining. During DF IEF, the selected protein bands have been concentrated up to 16.8-fold.
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Métodos Analíticos de la Preparación de la Muestra/métodos , Productos Biológicos/aislamiento & purificación , Focalización Isoeléctrica/métodos , Mezclas Anfólitas , Métodos Analíticos de la Preparación de la Muestra/instrumentación , Productos Biológicos/química , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/instrumentación , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Proteínas/química , Proteínas/aislamiento & purificación , Triticum/química , Levaduras/químicaRESUMEN
The instrument for continuous divergent flow IEF based on our principles set and outlined previously was further extended and tested. The separation and focusing area of a trapezoidal shape had a porous bed made from a nonwoven textile material with thickness decreasing from narrow input to wide output. A narrow end was used as a single input to continuously bring a single solution into separation space with a flow rate of 0.18 mL/min. Two pairs of electrodes were positioned close to both narrow and wide ends of the separation area with equilibrium state voltage of 75 V at the narrow input and 384 V at the wide output. Under dynamic equilibrium state, the zones of both pH gradient components and analytes were separated close to the input point and focused with increasing resolution while transporting through the separation space. The long-term stability experiments had shown the suitability of the device for preparative analysis; the zones of pI markers, hemoglobin and cytochrome C remained focused and separated over 15 h with deviations from the mean focusing positions ranging from 1.26 to 3.96% of the bed output width.
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Focalización Isoeléctrica/métodos , Proteínas/aislamiento & purificación , Citocromos c/aislamiento & purificación , Hemoglobinas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Poliésteres/química , Factores de TiempoRESUMEN
Studies in dynamic changes in protein translation require specialized methods. Here we examined changes in newly-synthesized proteins in response to ischemia and reperfusion using the isolated perfused mouse heart coupled with polysome profiling. To further understand the dynamic changes in protein translation, we characterized the mRNAs that were loaded with cytosolic ribosomes (polyribosomes or polysomes) and also recovered mitochondrial polysomes and compared mRNA and protein distribution in the high-efficiency fractions (numerous ribosomes attached to mRNA), low-efficiency (fewer ribosomes attached) which also included mitochondrial polysomes, and the non-translating fractions. miRNAs can also associate with mRNAs that are being translated, thereby reducing the efficiency of translation, we examined the distribution of miRNAs across the fractions. The distribution of mRNAs, miRNAs, and proteins was examined under basal perfused conditions, at the end of 30 min of global no-flow ischemia, and after 30 min of reperfusion. Here we present the methods used to accomplish this analysis-in particular, the approach to optimization of protein extraction from the sucrose gradient, as this has not been described before-and provide some representative results.