Electrophoretic Separation Of Proteins Research Articles

Effects of Effective Microorganisms on Meat Quality, Microstructure of the Longissimus Lumborum Muscle, and Electrophoretic Protein Separation in Pigs Fed on Different Diets.

Simple SummaryPork is the most popular meat among consumers in Poland, but it can also be a source of pathogens. Therefore, there is a need to find effective prophylaxis in order to ensure that consumers have access to safe food with the desired nutritional qualities and, above all, food that is free of pathogens. In order to meet these expectations, producers use probiotics containing effective microorganisms (EMs) offered in the market. In this study the same probiotic, EM Bokashi, was used for the first time in combination with three nutritional variants with proteins of different origin.The aim of the study was to determine how effective microorganisms influence meat quality, the microstructure of the longissimus lumborum muscle, and electrophoretic protein separation. The study group consisted of 150 piglets divided into three feeding groups: C, E1, and E2. The feeding groups included C—a standard fodder blend with a full share of post-extracted soya meal; E1—a 50%/50% mix of pea and lupine/soya bean in phase I of fattening and a 75%/25% mix of pea and lupine/soya bean in phase II of fattening; and E2—a 50%/50% mix of pea and lupine/soya bean in phase I of fattening and in 100% pea and lupine in phase II of fattening. The experimental factor was the addition of the EM Carbon Bokashi probiotic to the diet (C + EM, E1 + EM, E2 + EM). Influence of the feeding system on the following parameters was also estimated. After slaughter, the meat quality, LL muscle microstructure, and electrophoretic protein separation were assessed. In the C + EM group, a lower water-holding capacity was demonstrated. Meat from pigs fed the effective microorganism additive was much harder in the E1+EM group compared to meat from pigs from the E1 group. A beneficial effect of effective microorganism was found in the E2 + EM group, where less thermal leakage from the meat was demonstrated. A beneficial effect of the feeding system on thermal leakage and loin eye area in the E2 + EM group was demonstrated. In the C + EM group, a lower total number of muscle fibers was demonstrated. The addition of effective microorganism caused an increase in the diameter of fast twitch fibers in the E1 + EM group. In the same group of pigs, effective microorganisms caused a lower proportion of fiber fission. This nutritional variant appears to be the most appropriate for proteins as well, because it led to the most favorable percentage of individual proteins after effective microorganisms supplementation in the longissimus lumborum muscle.

Rapid Ultrasensitive Gel-Free Immunoblotting with Magnetic Labels

Immunoblotting is widely used for the detection of proteins using specific antibodies. We present here a new immunoblotting method, which is characterized by exceptional sensitivity, rapidness, and low consumption of antibodies. A thin conductive layer between touching hydrophilic cellulose membranes instead of polyacrylamide gel is used for the electrophoretic separation of proteins. Contrary to common Western blotting, the separation occurs in nondenaturing conditions. The membrane surface is smoothed by deposition of the cellulose layer and modified with azidophenyl groups, allowing for the photochemical in situ immobilization of proteins, which are carried out after the electrophoresis. Thus, the additional step of transferring the protein from the gel onto the membrane is eliminated. Specific protein bands are then visualized by decoration with magnetic beads. The limit of detection of interleukin IL-1β reaches 0.3 fg or ∼104 molecules, whereas the total blotting time is about 5 min. The application of the technique is demonstrated by the detection of IL-1β, total IgA, and IgA specific to Mycobacterium tuberculosis antigen in the exhaled breath samples, obtained from healthy subjects and tuberculosis patients.

Enhanced electrophoretic separation of proteins by tethered SiO2 nanoparticles in an SDS-polyacrylamide gel network.

Nanoparticles (NPs) are able to improve the separation efficiency of proteins in SDS-polyacrylamide gel electrophoresis (SDS-PAGE) due to their capability of enhancing heat dissipation during electrophoresis. However, the intrinsic surface charges of NPs (at buffer pH or charge induced due to the SDS coating) make them acquire electrophoretic mobility and movement in the gel. Such a movement leads to viscosity and temperature gradients in the gel and deteriorates the separation. In this work, we proposed a novel method by using tethered NPs in the gel. Silica NPs, as the model NPs, were prepared and their surfaces were modified using 3-[(methacryloxy)propyl] trimethoxysilane (MPS) which locks the NPs in the gel via covalent bonds (M-SiO2/PA (polyacrylamide)). SiO2 NPs were embedded into the gel (SiO2/PA) as the positive control, while pure PA gel was chosen as the negative control. The results showed that at a relatively high voltage of 250 V, although the Joule heat generated during electrophoresis disturbed the separation in the pure gel, the SiO2/PA and M-SiO2/PA nanocomposite gels showed better performances. In comparison with the pure PA gel, the resolution increased by 3 and 32% for SiO2/PA and M-SiO2/PA, respectively, in a relatively short separation time of 35 min. The gel with tethered NPs presented a more efficient separation in terms of band broadening and resolution compared with the gel with free NPs probably due to the movement of free charged particles in the gel. Evidently, the migration speed of protein bands in the gels decreased especially for larger proteins in the presence of the NPs compared to the pristine gel due to the steric hindrance of the NPs. Finally, we separated E. coli proteins, as a real sample. Among the three gels (pure PA, SiO2/PA, and M-SiO2/PA), the gel containing M-SiO2 showed the best performance.

Detection of Multisite Phosphorylation of Intrinsically Disordered Proteins Using Phos-tag SDS-PAGE.

Phos-tagTM SDS-PAGE is a method that enables electrophoretic separation of proteins based on their phosphorylation status. With Phos-tagTM SDS-PAGE, it is possible to discriminate between different phosphoforms of proteins based on their phosphorylation level and the number of phosphorylated sites, and to determine the stoichiometry of different phosphorylation products. Phos-tagTM SDS-PAGE is useful for analyzing disordered proteins with multiple phosphorylation sites and can be used for any of the downstream applications used in combination with conventional SDS-PAGE, for example, Western blotting and mass-spectrometry. To obtain the best results with Phos-tagTM SDS-PAGE, however, it is often necessary to optimize the gel composition. Depending on the molecular weight and number of phosphoryl groups added to the protein, different gel composition or running conditions should be used. Here, we provide protocols for Mn2+- and Zn2+-Phos-tagTM SDS-PAGE and give examples of how disordered proteins with different characteristics behave in gels with various Phos-tag concentrations.

Approval of the method of electrophoretic separation and identification of some urine proteins in rats with toxic nephropathy

The aim of the article. The aim was to test the method for determining certain protein markers in rat urine by electrophoretic separation followed by mass spectrometric identification for early diagnostic of nephrotoxicity. The objectives of the study included assessing the reproducibility of the protein separation method, comparing rats urine protein profiles normal and after gentamicin sulfate administration, as well as a biochemical and morphological study of kidney biopsy specimens.
 Materials and methods. Gentamicin sulfate (GS) was administered intramuscularly to rats of both sexes at a dose of 60 mg/kg b.w. for consequence 5 days. Daily urine was collected in the background, on the 3rd, 7th and 14th day from the start of the administration of GS in the metabolic cages. We measured the concentrations of total protein and creatinine in urine samples, the level of lipids in the homogenates of the kidneys. Electrophoretic separation of urine proteins was performed in the Mini-PROTEAN Tetra Vertical Electrophoresis. The gels were stained, and the stained zones were excised and enzymatic hydrolysis of the proteins was performed. Spectra of tryptic peptides were recorded on an ultrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker, Germany). Proteins were identified in the Biotools program by accessing the Mascot database (matrixscience.com).
 Results. Assessment of reproducibility of the results of electrophoretic separation of rat urine proteins showed an acceptable value of the coefficient of variation (on average 8.03%) and the error of analysis (analysis = 14.53%). It that normally in rats of males the alpha-2-uroglobulin precursor dominates in urine and in females immunoglobulin G light chains dominate was show. Against the background of the administration of GS, leukocytes in pathological concentrations appear in the urine in rats of both sexes; in males, the proteomic profile has a strong intraspecific dispersion, including due to sperm contamination; in females, well identified zones of albumin, alpha-1-antitrypsin, aminopeptidase M, beta-2-microglobulin, and transferrin was appear. A biochemical study of kidney homogenates an increase in total cholesterol (males) and triacylglycerides (females) revealed. Pathomorphological changes were similar in male and female rats in the form of fatty degeneration of the proximal tubule nephrothelium and leukocyte infiltration of interstitium and confirmed changes in the spectrum of urine proteins.
 Conclusion. Based on the analysis of the experimental results it was found that the use of the technology of electrophoretic separation of proteins in PAGE under denaturing conditions followed by MALDI mass spectrometric identification (PAGE-MALDI-TOF/TOF) and densitometric determination of the percentage of protein fractions is applicable for the detection of nephrotoxicity. Due to gender differences in the protein spectrum it is preferable to examine the urine of female rats. Pathomorphological changes did not have gender differences.

Influence of medication “Membranostabil” on expression of immunoreceptor proteins in small intestine of ruminants during the period of formation of colostral immunity

The influence of medication “Membranostabil” on the indexes of content of proteins with MM of 33, 40, and 47 kDa in the plasmolemma of enterocytes of jejunum of new-born calves during the period from birth till the age of 24-hours was investigated. The research was conducted on calves (males) of Ukrainian black-and-white breed during the spring. Throughout the trial period, the calves of the control and experimental groups were kept on a standard diet for newborn calves, colostrum. In addition, calves of the experimental group got the medication “Membranostabil” based on the soybean lecithin. Proteins of plasmolemma of enterocytes of the jejunum of calves were isolated from the membrane fraction of lysates and examined by electrophoresis. Electrophoretic separation of proteins of plasmolemma of the jejunum enterocytes of newborn calves revealed proteins with MM of 37, 40 and 43 kDa on electrophoregram. The data of expression of immunoreceptor proteins with MM of 37, 40, 43 kDa and the dynamics of their changes depending on the time after birth in calves of the control and experimental groups were analyzed. The decrease in the total content of expression of proteins with MM of 37, 40, and 43 kDa in the membrane fraction of lysates of enterocytes of the jejunum of calves at the age of 6 hours compared to the newborn ones was demonstrated. The levels of expression of each immunoreceptor protein are considered with their comparative analysis between the individual fractions in the cytolemma of the enterocytes of the jejunum of newborn calves, which according to their molecular weights correspond to MM isoforms of FcγRIII/CD32. The data obtained showed a significantly higher expression of level of proteins with molecular mass of 37 kDa on the apical membrane of enterocytes, compared with proteins with MM of 40 and 43 kDa, both at birth of calves and at the age of 6 and 24 hours. Based on the data obtained, proteins with MM of 37, 40, and 43 kDa of plasmolemma of the jejunum enterocytes undergo significant changes in the process of postnatal ontogeny in cattle. These changes indicate the synthesis of these proteins in the enterocytes themselves in the process of formation of colostral immunity in newborn calves under the influence of the medication “Membranostabil” used by this research group, compared with calves of the control group. It is shown that the application of medication “Membranostabil” for the new-born calves activates the transport of immunoglobulins of colostrum in the intestine of these animals, assists the increasing of level of colostral immunity, and prevents the occurrence of digestive disorders. Findings of this research correlate with the results of clinical observation in newborn calves.

Izozyme polymorphism of alpha-amylase in soft wheat

Aim. Study of alpha amylase polymorphism in winter wheat varieties. Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The fall number on the Falling Number 1700 was determined. Results. Strains from various collections were explored: Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kiev, The Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation of the National Academy of Agricultural Sciences of Ukraine, Odesa and german strains. The polymorphism of wheat grain alpha-amylase was detected by the method of electrophoretic protein separation. We searched associations between variants of alpha-amylase and resistance to pre-harvest germination, the falling number in the examined samples was determined. Conclusions. The frequency of the prevalence of variants for isoenzymes of alpha-amylase in soft wheat varieties was estimated. It was shown that genotypes containing the variant of the isoenzymes АbC are more resistant to pre-harvest germination. Among the studied varieties of PBGI NAAS and IPPG NAS of Ukraine, the variant of alpha-amylase AbC.
 Keywords: α-amylase, isoenzymes, soft wheat, electrophoresis, falling number.

Quantification of In Vivo Target Engagement Using Microfluidic Activity-Based Protein Profiling.

Accurate measurement of drug-target interactions in vivo is critical for both preclinical development and translation to clinical studies, yet many assays rely on indirect measures such as biomarkers associated with target activity. Activity-based protein profiling (ABPP) is a direct method of quantifying enzyme activity using active site-targeted small-molecule covalent probes that selectively label active but not inhibitor-bound enzymes. Probe-labeled enzymes in complex proteomes are separated by polyacrylamide gel electrophoresis and quantified by fluorescence imaging. To accelerate workflows and avoid imaging artifacts that make conventional gels challenging to quantify, we adapted protocols for a commercial LabChip GXII microfluidic instrument to permit electrophoretic separation of probe-labeled proteins in tissue lysates and plasma, and quantification of fluorescence (probe/protein labeling ratio of 1:1). Electrophoretic separation on chips occurred in 40 s per sample, and instrument software automatically identified and quantified peaks, resulting in an overall time savings of 3-5 h per 96-well sample plate. Calculated percent inhibition was not significantly different between the two formats. Chip performance was consistent between chips and sample replicates. Conventional gel imaging was more sensitive but required five times higher sample volume than microfluidic chips. Microfluidic chips produced results comparable to those of gels but with much lower sample consumption, facilitating assay miniaturization for scarce biological samples. The time savings afforded by microfluidic electrophoresis and automatic quantification has allowed us to incorporate microfluidic ABPP early in the drug discovery workflow, enabling routine assessments of tissue distribution and engagement of targets and off-targets in vivo.

Heat dissipation in slab gel electrophoresis: The effect of embedded TiO2 nanoparticles on the thermal profiles

Despite the fast development of novel and high-resolution electrophoresis techniques such as capillary-based methods and microfluidic devices, the slab gel electrophoresis is still a popular method for the separation of biomolecules in medicine and biology. It is a low cost and simple method and offers high throughput. However, this technique is limited to low voltages leading to slow separations. Producing the heat during the electrophoresis known as Joule heating inevitably leads to a rise in the gel temperature. For the first time, this work offers a whole gel temperature measurement by using a thermal camera which presents accurate temperature profiles in the gel with a resolution of more than 10 pixel/mm2 and a precision of 0.1 °C. Titania, TiO2, nanoparticles (NPs) were embedded into the polyacrylamide (PA) gel to improve the electrophoretic separation of proteins. By embedding 0.025% w/v TiO2 NPs, heat dissipation increases by 16.5% at applied voltage of 200 V compared with that of PA gel with no embedded TiO2 NPs. The thermal images showed that the composite gel was 2.5 °C in average cooler than PA gel after 15 min of electrophoresis run at 200 V. The maximum separation voltage increased by 30 V in the composite PA/TiO2 gel compared with the pure PA gel. Moreover, the average number of theoretical plates over the 10 protein peaks, as a criterion of separation performance, increased by about 63% at 180 V when TiO2 NPs were included into the gel.

Polyurethane Foam in a Reliable Method for Electrophoretic Separation of Proteins

Polyurethane Foam in a Reliable Method for Electrophoretic Separation of Proteins

Electrophoretic Separation of Very Large Molecular Weight Proteins in SDS Agarose.

Very large proteins (subunit sizes, >200kDa) are difficult to electrophoretically separate on polyacrylamide gels. A SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. Proteins with molecular masses between 200 and 4000kDa can be clearly separated. Inclusion of a reducing agent in the upper reservoir buffer and use of a large pore-sized agarose have been found to be key technical procedures for obtaining optimum protein migration and resolution.

Isoenzymes spectrum of genes expression of cattle in different directions of productivity

While studying the peculiarities of the species of genetic structure of cattle, depending on the direction of productivity, one can predict, that there may be certain mechanisms that have an intermediate role between the genetically determined polymorphism of biochemical markers and the variability of complexity of the signs of productivity. One of such mechanism may be the variability of the “biochemical phenotype” of different organs – a number of organ-specific isoenzymetric spectra of genetic-biochemical systems. In this regard, in this experiment organ-specific peculiarities of the isoenzyme spectrum of various enzymes in animals of dairy and meat production were considered. For this experiment, samples of meat, and then dairy animals in pairs were placed in the electrophoretic block in the following order: lungs, heart muscle, spleen, skeletal muscle and liver. The features of the organ-specific isoenzyme spectrum of the enzymes of the general intracellular metabolism are breed and species specific. Therefore, a comparative analysis of the organ-specific spectrum of isoenzymes of various genetic-biochemical systems in these studies was performed on a small number of animals (3–5 heads). The features of the isoenzyme spectrum were evaluated by genetically-determined polymorphisms of groups of genetic-biochemical systems. Experiments were carried out on samples of homogenates of organs obtained with the addition of trilon-B. Polymorphism of enzymes was evaluated using a method of electrophoretic protein separation in 13% starch gel in horizontal chambers with subsequent histochemical staining. These searches indicate the presence of pronounced organospecificity of the isoenzyme spectrum of purinucleoside phosphorylase, malate dehydrogenase, malic enzyme, and lactate dehydrogenase in cattle. The intraspecific differences of the organ-specific isoenzyme spectrum were revealed. It has been shown that the expression of the genetic and biochemical systems under investigation is significantly different in cattle of dairy and meat production lines in the cardiac and skeletal muscles. The “biochemical phenotype” of the heart muscle and skeletal muscle of dairy cattle is significantly different from the livestock of the meat production direction. Such studies may lead to the identification of characteristic genotypes in a complex of genetic-biochemical systems, which are closely related to the corresponding complex of economic benefits.

Effect of Cystoseira barbata and Dictyota dichotoma-algae on reproduction and protein pattern of Biomphalaria alexandrina snails

ABSTRACTControlling the snail intermediate host of schistosomiasis is one of the strategies used to reduce disease incidence. In this study screening tests were carried out on the action of dried powder of eight algal species against Biomphalaria alexandrina snails. The most effective powders were from Dictyota dichotoma and Cystoseira barbata with LC50 values of 560 and 2200 ppm, respectively. Prolonged exposure to sublethal concentrations of these two powders caused adverse effects on snails' reproduction especially at LC25. Hatchability of exposed eggs was lowest at LC25 of C. barbata, and LC15 of D. dichotoma. The most pronounced effects on the hermaphrodite gland of snails were caused by high concentrations of both algal suspensions, where ova were either degenerated or deformed and there were detrimental effects on all spermatogenesis stages. The electrophoretic separation of haemolymph proteins showed that the concentration with most effect was the LC15 for both tested algae as they resulted in more bands than the control and also an elevation in total protein content.

Polymorphism of alpha-amylase of soft wheat

Aim. Study of polymorphism of alpha amylase in winter wheat varieties. Methods. Isoenzymes of alpha-amylase were detected by electrophoretic protein separation in a polyacrylamide gel. The germination index (GI) was calculated by Walker-Simmons: GI = (7xn1 + 6xn2 + ... + 1xn7) / 7 x N, where n1, n2, ..., n7 is the number of seeds sprouted on the first, second, and further days to the seventh day, respectively, N – the total number of grains. Results. Varieties were explored: Institute of Plant Physiology and Genetics of the National Academy of Sciences of Ukraine, Kiev, The Plant Breeding and Genetics Institute – National Center of Seed and Cultivar Investigation of the National Academy of Agricultural Sciences of Ukraine, Odesa and german varieties.The polymorphism of wheat grain alpha-amylase was detected by the method of electrophoretic protein separation. We searched associations between variants of alpha-amylase and resistance to pre-harvest germination, the germination index in the studied samples was determined. Conclusions. The frequency of the prevalence of variants for isoenzymes of alpha-amylase in soft wheat varieties was estimated. It was shown that genotypes containing the variant of the isoenzymes АbC are more resistant to pre-harvest germination. Among the studied varieties of PBGI NAAS and IPPG NAS of Ukraine, the variant of alpha-amylase AbC.
 Keywords: α-amylase, soft wheat, electrophoresis, varieties.

Cytomorphological Indicators of Lymphoid Tissue and Protein Composition of Cattle Blood during the Prenatal and Postnatal Periods

The cytomorphological reactions of lymphoid tissue and the immunobiological status of the cattle organism in the prenatal and postnatal periods were analyzed. The cytomorphological analysis was carried out by counting the lymphoid and plasma cells in 50 fields of vision according to the method in [10]. The level of metabolic processes in the experimental animals was determined by the content of the total protein in their blood serum estimated by refractometry [11]. Protein fractions were described with the electrophoresis in agar gel according to [12]. The immunoelectrophoresis was performed as described in [13]. The main shifts in the cellular composition of the lymph nodes and spleen, as well as other lymphoid tissues in early fetuses, were expressed in the accumulation of lymphoblasts, which were large basophilic cells. Electrophoretic separation and immune-based visualization of experimental animals' serum proteins were carried out simultaneously, and the phenomenon of complete antigenic identity was observed with both immune sera against cows’ and bulls’ blood proteins. From 18 to 22 precipitates were detected on their immunophoregrams.

Options for genetic-biochemical markers in connection with dairy productivity

The purposeful creation and use of highly productive animals largely depends on information laid down in their genes that control valuable economic useful features. The actual task is to identify and use marker genes that are responsible for displaying a certain sign of animals, in particular, the level of milk productivity. To create new forms of animals with a combination of high productivity and adaptability to various factors of external influences, for this purpose, for a long time in Ukraine, work was carried out to improve the Simmental cattle using the Red-Spoptted Holstein breed. It was supposed to that the increase in the number of animals with a part of the heredity of Red-Spoptted Holstein to 70–80% will allow to receive herds, which are characterized by good adaptive capacity, fat-dairy, high growth and meat qualities, as well as high milk yield. The searches were carried out on cows (pure-bred Simmentals and between ½ and ¾ in terms of the heredity of the Red-Spoptted Holstein breed), which were divided into groups with different levels of milk productivity for the first lactation (on the milk yield and content of fat in milk). The genetic structure was estimated by genetically determined polymorphism of groups of genetic-biochemical systems. Experiments were conducted on erythrocytes and blood plasma.Blood from animals was taken from a jugular vein in a heparin test tube. Polymorphism of proteins and enzymes was evaluated using a method of electrophoretic protein separation in 13% starch gel in horizontal chambers followed by histochemical staining. The obtained data testify that differences of animals on such parameters of dairy as milk yield for 305 days of the first lactation and the content of fat in milk is accompanied by unequal differentiation of their genetic structures by different genetic-biochemical systems. With the difference in milk yield, the most pronounced changes are observed with the transferrin locus, and in the analysis of groups of animals differing in the fat content of milk – by the AM-1 locus. This suggests that, based on such cases of negative correlations between these features, specific interlocal interactions may be based on different genetic and biochemical systems. It can be argued that the development of such searches may lead to the discovery of characteristic genotypes by a complex of genetic-biochemical systems, closely related to the corresponding complex of economic useful features.

Detection and molecular characterization of sorbitol fermenting non-O157 Escherichia coli from goats

Shiga toxigenic Escherichia coli (STEC) O157 and several other serogroups of non-O157 STEC strains present as commensalism bacteria in small ruminants that possess a high potential of attaining pathogenic virulent genetic elements. The pathogenicity of non-O157 strains is emerging progressively in animals as well as in humans especially in the rural areas as they get transmitted through unsanitary practices of living, consumption of uncooked meat and milk, human-livestock close proximity as well as within livestock pathogenic bacterial transmission. The present study was carried out to determine the prevalence of non-O157 E.coli isolates and characterize based on clinical history, antibiotic sensitivity testing, multiplex PCR (mPCR) detection of virulence genes, genotype identification using pulse field gel electrophoresis (PFGE) and polyacrylamide gel electrophoretic separation of antigenic proteins to determine their prevalence, virulence and genetic comparison between host and environment for epidemiological significance. A total of 300 E.coli isolates were recovered from rectal swabs of goats and their surrounding environment over a period of one year (2016–2017) by selective isolation. Among which 50 isolates were confirmed to be from the non-O157 E.coli family. The mPCR analysis of these 50 isolates revealed the presence of two or more virulent genes, viz., hylA (90%), fliC (74%), eaeA (56%), stx1 (48%) and stx2 (22%).Four isolates exhibited multidrug-resistance to amoxiclav, doxycycline, ciprofloxacin and ceftriazone. The PFGE fingerprint profile showed six major clusters at 100% similarity from the 50 isolates. The major antigenic proteins identified from the isolates were stx1A, stx2B and fliC. This study has significant implications for understanding the molecular diversity of emerging pathotypes of non-O157 in young goats in terms of virulence and epidemiological aspects.

Mouse-to-mouse variation in maturation heterogeneity of smooth muscle cells.

Smooth muscle cell (SMC) heterogeneity plays an important role in vascular remodeling, a life-threatening hallmark of many vascular diseases. However, the characterization of SMCs at the single-cell level is stymied by drawbacks of contemporary single-cell protein measurements, including antibody probe cross-reactivity, chemical fixation artifacts, limited isoform-specific probes, low multiplexing and difficulty sampling cells with irregular morphologies. To scrutinize healthy vessels for subpopulations of SMCs with proliferative-like phenotypes, we developed a high-specificity, multiplexed single-cell immunoblotting cytometry tool for unfixed, uncultured primary cells. We applied our assay to demonstrate maturation stage profiling of aortic SMCs freshly isolated from individual mice. After ensuring unbiased sampling of SMCs (80-120 μm in length), we performed single-SMC electrophoretic protein separations, which resolve protein signal from off-target antibody binding, and immunoblotted for differentiation markers α-SMA, CNN-1 and SMMHC (targets ranging from 34 kDa to 227 kDa). We identified a subpopulation of immature-like SMCs, supporting the recently-established mechanism that only a subset of SMCs is responsible for vascular remodeling. Furthermore, the low sample requirements of our assay enable single-mouse resolution studies, which minimizes animal sacrifice and experimental costs while reporting animal-to-animal phenotypic variation, essential for achieving reproducibility and surmounting the drawbacks of pooling primary cells from different animals.

Utilization of dried and long-term stored polyacrylamide gels for the advanced proteomic profiling of mitochondrial contact sites from rat liver.

Following subcellular fractionation, the complexity of proteins derived from a particular cellular compartment is often evaluated by gel electrophoretic analysis. For the proteomic cataloguing of these distinct protein populations and their biochemical characterization, gel electrophoretic protein separation can be conveniently combined with liquid chromatography mass spectrometry. Here we describe a gel-enhanced liquid chromatography mass spectrometry (GeLC-MS)/MS approach with a new bioanalytical focus on the proteomic profiling of mitochondrial contact sites from rat liver using the highly sensitive Orbitrap Fusion Tribrid mass spectrometer for optimum protein identification following extraction from dried and long-term stored gels. Mass spectrometric analysis identified 964 protein species in the mitochondrial contact site fraction, whereby 459 proteins were identified by ≥3 unique peptides. This included mitochondrial components of the supramolecular complexes that form the ATP synthase, the respiratory chain, ribosomal subunits and the cytochrome P450 system, as well as crucial components of the translocase complexes translocase of the inner membrane (TIM) and translocase of the outer membrane (TOM) of the two mitochondrial membranes. Proteomics also identified contact site markers, such as glutathione transferase, monoamine oxidase and the pore protein voltage dependent anion channel (VDAC)-1. Hence, this report demonstrates that the GeLC-MS/MS method can be used to study complex mixtures of proteins that have been embedded and stored in dried polyacrylamide gels for a long period of time. Careful re-swelling and standard in-gel digestion is suitable to produce peptide profiles from old gels that can be used to extract sophisticated proteomic maps and enable the subsequent bioinformatics analysis of the distribution of protein function and the determination of potential protein clustering within the contact site system.

New Approach to the Formation of Physically Adsorbed Capillary Coatings Consisting of Hyperbranched Poly(Ethylene Imine) with a Maltose Shell to Enhance the Separation of Catecholamines and Proteins in CE

Core–shell-type polymers based on a hyperbranched (hb) poly(ethylenimine) core and a shell with a variable maltose content were applied as coating materials for fused silica capillaries. A new, simple, fast, and reproducible way of modifying the capillary walls through the physical adsorption of the core–shell-type polymers using a Cu2+ support was developed. The coating created by this method was found to be very stable compared to the coating created using a solution of the polymer only. Capillaries modified with the core–shell-type polymers were tested by applying them to the electrophoretic separation of catecholamines and proteins. The modified capillaries showed high efficiencies (up to 800,000 theoretical plates per meter for lysozyme) and separation selectivities. The highest efficiency was achieved using capillaries modified with the polymer containing the lowest content of maltose in the shell and the most accessible positively charged core. Various online concentration techniques were also tested as a means to lower detection limits further, making it possible to analyze proteins in biological fluids (saliva) as well as catecholamines in human urine after SPE using activated alumina.