One-dimensional Electrophoresis Research Articles

Stress Adaptation Responses of a Listeria monocytogenes 1/2a Strain via Proteome Profiling.

Listeria monocytogenes is a foodborne pathogen that is ubiquitous and largely distributed in food manufacturing environments. It is responsible for listeriosis, a disease that can lead to significant morbidity and fatality in immunocompromised patients, pregnant women, and newborns. Few reports have been published about proteome adaptation when L. monocytogenes is cultivated in stress conditions. In this study, we applied one-dimensional electrophoresis and 2D-PAGE combined with tandem mass spectrometry to evaluate proteome profiling in the following conditions: mild acid, low temperature, and high NaCl concentration. The total proteome was analyzed, also considering the case of normal growth-supporting conditions. A total of 1,160 proteins were identified and those related to pathogenesis and stress response pathways were analyzed. The proteins involved in the expression of virulent pathways when L. monocytogenes ST7 strain was grown under different stress conditions were described. Certain proteins, particularly those involved in the pathogenesis pathway, such as Listeriolysin regulatory protein and Internalin A, were only found when the strain was grown under specific stress conditions. Studying how L. monocytogenes adapts to stress can help to control its growth in food, reducing the risk for consumers.

Characterisation of the Theileria orientalis Piroplasm Proteome across Three Common Genotypes.

Theileria orientalis is an emerging apicomplexan pathogen of cattle occurring in areas populated by the principal vector tick, Haemaphysalis longicornis. Unlike transforming Theileria spp. that induce cancer-like proliferation of lymphocytes via their schizont stage, T. orientalis destroys host erythrocytes during its piroplasm phase resulting in anaemia. The underlying pathogenic processes of T. orientalis infection are poorly understood; consequently, there are no vaccines for prevention of T. orientalis infection and chemotherapeutic options are limited. To identify antigens expressed during the piroplasm phase of T. orientalis, including those which may be useful targets for future therapeutic development, we examined the proteome across three common genotypes of the parasite (Ikeda, Chitose and Buffeli) using preparations of piroplasms purified from bovine blood. A combination of Triton X-114 extraction, one-dimensional electrophoresis and LC-MS/MS identified a total of 1113 proteins across all genotypes, with less than 3% of these representing host-derived proteins. Just over three quarters of T. orientalis proteins (78%) identified were from the aqueous phase of the TX-114 extraction representing cytosolic proteins, with the remaining 22% from the detergent phase, representing membrane-associated proteins. All enzymes involved in glycolysis were expressed, suggesting that this is the major metabolic pathway used during the T. orientalis piroplasm phase. Proteins involved in binding and breakdown of haemoglobin were also identified, suggesting that T. orientalis uses haemoglobin as a source of amino acids. A number of proteins involved in host cell interaction were also identified which may be suitable targets for the development of chemotherapeutics or vaccines.

Comparative proteomic study of pig muscle proteins during growth and development of an animal

The production of high-quality pork is closely related to the growth and development of muscle tissue. The present article provides a comparative proteomic research of l. dorsi, b. femoris, m. brachiocephalicus during the pigs’ growth and development (at age of 60 days and 180 days). This work was supported by data of electrophoretic methods: one-dimensional electrophoresis according to Laemmli with densitometric assessment in the ImageJ software and two-dimensional electrophoresis according to O’Farrell method with its further processing on the software ImageMaster. The mass spectrometric identification was conducted with the help of the high-performance liquid chromatography (HPLC) system connected to a mass spectrometer; further the data were interpreted by search algorithm Andromeda. When comparing frequency diagrams of one-dimensional electrophoregrams of all three muscle tissues of weaned pigs, the greatest difference was observed for the muscle sample l. dorsi. Comparison of diagrams of muscle tissue samples taken for mature pigs showed a great similarity of all three studied muscles samples. Within the framework of the research, the Fold indicator was calculated. The exceeding its value by more than 2 units is generally considered to be a statistically significant difference. When analyzing two-dimensional electrophoretograms of weaned pigs’ muscles, 18 protein fractions were revealed with Fold > 2. When examining the muscle tissue of mature pigs, 15 of those proteins were found; the differences were mostly detected in the minor protein fractions. The mass spectrometric analysis of the cut bands with well-pronounced differences from the onedimensional electrophoretogram revealed 214 proteins involved to a greater extent in cellular and metabolic processes, physical activity and localization. Growth and development protein — semaphorin‑6B (96.78 kDa) — was revealed in muscle tissue of l. dorsi, a. Also in l. dorsi and b. femoris the growth and development proteins were found: cadherin‑13 (78.23 kDa), cadherin‑7 (87.01 kDa), the F‑actin-cap protein beta subunit (30.66 kDa), and two uncharacterized proteins at 65.60 kDa and 63.88 kDa.

From birth to adulthood: An analysis of the Brazilian lancehead (Bothrops moojeni) venom at different life stages.

The Brazilian lancehead (Bothrops moojeni) has a wide distribution in Brazil and represents a serious public health hazard. Previous works reported that the symptoms of snakebites caused by B. moojeni juveniles' bites were mainly related to coagulation, while those caused by adults' bites had a more prominent local damage. In this work, we analyzed the venoms of B. moojeni at different life stages to better understand the ontogeny shift in this species. Snakes were grouped by age and sex, and venom pools were formed accordingly. Compositional analyses by one-dimensional electrophoresis (1-DE), chromatography, and mass spectrometry revealed that ontogenetic changes might be mostly related to phospholipase A2 (PLA2) and metalloproteases. Regarding the venoms functional aspect, proteolytic, L-amino acid oxidase, PLA2, and coagulant in vitro activities were assayed, but only the first and the last ones showed age-related changes, with the venom of snakes up to 1 year-old displaying lower proteolytic and higher coagulant activities, while those from 2 years-old onward presented the opposite relation. The venoms of 3 years-old snakes were exceptions to the compositional and functional pattern of adults as both venoms presented profiles similar to neonates. Sex-related differences were observed in specific groups and were not age-related. In vivo experiments (median lethal dose and hemorrhagic activity) were statistically similar between neonates and adults, however we verified that the adult venom killed mice faster comparing to the neonates. All venoms were mostly recognized by the antibothropic serum and displayed similar profiles to 1-DE in western blotting. In conclusion, the Brazilian lancehead venom showed ontogenetic shift in its composition and activities. Furthermore, this change occurred in snakes from 1 to 2 years-old, and interestingly the venom pools from 3 years-old snakes had particular characteristics, which highlights the importance of comprehensive studies to better understand venom variability.

Molecular Characterization of Secreted Proteins from Salivary Gland Immunogenic Protein of Anopheles vagus

Salivary mosquito’s proteins have been widely acknowledged to contain factors important for pathogen transmission as well as for biomarker of Anopheles exposure. The objective of this study was to identify and characterize the secreted proteins from salivary glands and immunogenic proteins from of Anopheles (An.) vagus. A proteomic approach combining one-dimensional electrophoresis (1DE) was operationalized, followed by western blot analysis using human sera from healthy people living in an endemic area (Kendal, East Java - Indonesia); liquid chromatography mass spectrometry (LC-MS/MS) and bioinformatics analysis were conducted to gain direct insight into An. vagus salivary proteins. Three immunogenic proteins with molecular weight of 69, 75 and 232 kDa were identified. Apart from the housekeeping proteins identified by LC-MS/MS, there were also some proteins which played crucial role in the blood feeding process i.e AGA 5’ nucleotidase family, for 69 & 75 kDa and SGS 4 for 232 kDa respectively. The other known proteins like vitellogenin, putative myosin class I heavy chain and heat shock protein 70 (Hsp70) were also identified. The majority of proteins were clearly characterized in Anopheles for their role in blood feeding, metabolism, cytoskeleton protein. and stress response.

Genetic determinants of seed protein plasticity in response to the environment in Medicago truncatula.

As the frequency of extreme environmental events is expected to increase with climate change, identifying candidate genes for stabilizing the protein composition of legume seeds or optimizing this in a given environment is increasingly important. To elucidate the genetic determinants of seed protein plasticity, major seed proteins from 200 ecotypes of Medicago truncatula grown in four contrasting environments were quantified after one-dimensional electrophoresis. The plasticity index of these proteins was recorded for each genotype as the slope of Finlay and Wilkinson's regression and then used for genome-wide association studies (GWASs), enabling the identification of candidate genes for determining this plasticity. This list was enriched in genes related to transcription, DNA repair and signal transduction, with many of them being stress responsive. Other over-represented genes were related to sulfur and aspartate family pathways leading to the synthesis of the nutritionally essential amino acids methionine and lysine. By placing these genes in metabolic pathways, and using a M.truncatula mutant impaired in regenerating methionine from S-methylmethionine, we discovered that methionine recycling pathways are major contributors to globulin composition establishment and plasticity. These data provide a unique resource of genes that can be targeted to mitigate negative impacts of environmental stresses on seed protein composition.

Identification of conserved Mycoplasma agalactiae surface antigens by immunoproteomics

Contagious agalactia represents one of the most relevant infectious diseases of dairy sheep, with Mycoplasma agalactiae being the primary etiological agent. The early, sensitive, and specific identification of infected animals, as well as the development of efficient prophylactic tools, remain challenging. Here, we present a comprehensive characterization of M. agalactiae antigens focusing on those shared among different isolates. Leveraging on previous proteomic data obtained on individual strains, we adopted a strategy entailing sample pooling to optimize the identification of conserved proteins that induce an immune response. The liposoluble proteins from previously characterized field isolates and the type strain PG2T were enriched by Triton X-114 fractionation, pooled, analysed by one-dimensional (1D) and two-dimensional (2D) electrophoresis, and subjected to western immunoblotting against sheep sera collected during natural infection with M. agalactiae. Immunodominant antigens were identified by Matrix-Assisted Laser Desorption-Time-Of-Flight-Mass Spectrometry (MALDI-TOF-MS). This combined immunoproteomic approach confirmed the role of several known immunogens, including P80, P48, and P40, and most variable surface proteins (Vpmas), and unveiled novel immunodominant, conserved antigens, including MAG_1000, MAG_2220, MAG_1980, phnD, MAG_4740, and MAG_2430. Genomic context, functional prediction, subcellular localization, and invariable expression of these proteins in all isolates suggest their possible involvement in bacterial pathogenicity and metabolism. Moreover, most of the identified antigens elicit a host humoral response since the early stages of infection, persisting for at least 270 days. The immunodominant, conserved antigen panel identified in this work supports the development of effective vaccines and diagnostic tools with higher sensitivity and specificity in all the natural infection stages.

Immunoproteomic Analysis of Dirofilaria repens Microfilariae and Adult Parasite Stages.

Dirofilariarepens is a parasitic nematode causing a vector-borne zoonotic infection (dirofilariosis), considered an emerging problem in human and veterinary medicine. Currently, diagnosis is based on the detection of the adult parasite and microfilariae in the host tissues. However, the efficacy of tests relying on microfilariae detection is limited by microfilariae periodic occurrence. Therefore, a new reliable and affordable serological diagnostic method is needed. Better characteristic of the parasite biology and its interaction with host immune system should help to achieve this goal. This study analyzes adult and microfilariae proteomes, and the use of one-dimensional electrophoresis (1-DE) and two-dimensional electrophoresis (2-DE) proteomics, immunoproteomics, and LC-MS/MS mass spectrometry allowed us to identify 316 potentially immunogenic proteins (75 belong to adult stage, 183 to microfilariae, and 58 are common for both). Classified by their ontology, the proteins showed important similarities and differences between both parasite stages. The most frequently identified proteins are structural, metabolic, and heat shock proteins. Additionally, real-time PCR analysis of some immunogenic targets revealed significant differences between microfilariae and adult life stages. We indicated molecules involved in parasite-host interactions and discussed their importance in parasite biology, which may help to reveal potential diagnostic antigens or select drug and vaccine targets.

Development of a Method for the Extraction of the Total Proteome of Bacillus anthracis Spores

We have developed a method for obtaining the total proteome of <i>Bacillus anthracis</i> spores, which combines efficient protein extraction with reliable disinfection of samples. We used 7 strains of <i>B. anthracis</i>: 4 plasmid-free, 3 diplasmid, one of which with an atypical type of capsule formation in air. The schemes for isolating the total spore proteome were tested using various lysis solutions in the presence of bacterial protease inhibitors, with the inclusion of the stages of treatment of spores with trichloroacetic acid and mechanical disintegration and with their exclusion. The quality and completeness of the extraction of the total proteome of the spores of the samples was assessed in one-dimensional (1D) and twodimensional (2D) electrophoresis. Treatment of spores with trichloroacetic acid increased the reliability of material disinfection and reduces the loss of the final product. Mechanical disintegration after treatment of spores with lysing solutions increases the completeness of extraction of spore proteins in a wide range of molecular weights and facilitated the process of sterilizing filtration. Final filtration of the lysate through a PVDF filter with a pore size of 0.22 μm provided additional decontamination of samples without reducing their quality. Thus, the use of the proposed sample preparation scheme makes it possible to obtain complete protein extracts of spores of <i>B. anthracis</i> strains, suitable for comparative analysis of the proteome and search for a possible correlation with the features phenotypic properties and mechanisms that ensure the preservation of the pathogen anthrax in environmental objects, including soil

Identification of Proteins Associated with the Formation of Oral Biofilms

Objective: To identify proteins associated with the formation of Streptococcus gordonii and Fusobacterium nucleatum biofilms. Material and Methods: Biofilms composed of two bacterial species, S. gordonii and F. nucleatum, were cultured for 1, 4, 7, and 10 days. The presence of both species was confirmed via amplification of the srtA and radD genes using real-time PCR. The concentrations of proteins associated with the biofilms and individual species were quantified using Western blotting. Results: The protein profiles of S. gordonii and F. nucleatum from individual cultures determined using one-dimensional electrophoresis revealed proteins found in S. gordonii and in F. nucleatum. Ct and reciprocal Ct values were determined for the exposed S. gordonii and F. nucleatum biofilms. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein was detected in biofilms and F. nucleatum, whereas HSP40 protein was present only in biofilms after 7 and 10 days of formation. Conclusion: HSP40 was detected only in the formed biofilms; thus, HSP40 is an essential proteins for adhesion.

Protein profile as a quality indicator of cryopreserved semen from Tambaqui Colossoma macropomum (Cuvier, 1818).

The aim of this study was to evaluate the association between proteins in the seminal plasma of tambaqui Colossoma macropomum (Cuvier, 1818) with seminal quality indicators after thawing. The semen was cryopreserved with a dilution based on BTS with 8% DMSO. A 200 µL sample of semen from each animal was diluted in 800 µL BTS, centrifuged at 800 rpm, and the supernatant was cryopreserved to further analyze of the protein profile of seminal plasma through one-dimensional electrophoresis (SDS-PAGE). After 15 days of cryopreservation, a cryopreserved semen straw was thawed to analyze both qualitative and quantitative parameters. When considering all collections, the SDS-PAGE identified 15 protein bands in the seminal plasma of tambaqui. When the interaction (presence or absence) between proteins observed in the seminal plasma and the post thawed spermatic parameters was evaluated, we observed a great influence of the presence of proteins on spermatic quality. A greater (P<0.05) fertilization rate was observed with the presence of proteins 12, 34, 44, 85, and 90 kDa. Proteins in seminal plasma of tambaqui influenced the spermatic quality after thawing, and thus, they can be utilized as an indicator of sperm quality, especially the proteins with a molecular weight ≤ 50 kDa.

Study of the functional product’s protein compounds digestion features

The aim of the study was to investigate the transformation of meat product’s proteins from pig hearts and aortas during enzymatic hydrolysis in an in vitro model of the gastrointestinal tract. The model consisted of three phases simulating digestion processes: “oral cavity” phase (a-amylase, pH 7.0; 2 min), “stomach” phase (pork pepsin, pH 3.0; 120 min), “intestine” phase (pork pancreatin, pH 7.0; 130 min). The product was sequentially subjected to hydrolysis, at the end of each phase, samples were taken to determine the protein concentration (biuret method) and visualize the protein fractions (one-dimensional electrophoresis). A significant increase in protein concentration at the “stomach” phase was revealed by 3.2 times, and the absolute content by 4.6 times. At the “intestine” phase, a decrease in the number of peptide complexes with copper ions by 1.8 times, the absolute protein content by 8.5% was re‑ vealed. The noted tendency was confirmed by electrophoretic studies — at the stage, simulating digestion in the stomach, the prod‑ ucts of meat product’s proteins hydrolysis were visualized; at the “intestine” phase, a low expression of protein fractions in the range of more than 10 kDa is shown. The maximum hydrolysis of protein compounds at the “stomach” phase to poly- and oligopeptides was confirmed, continuing at the “intestine” stage with the accumulation of free amino acids. This methodology makes it possible to visualize the products of hydrolysis of proteins in a meat product at all stages of the model and to monitor changes in protein concentration in the system.

Smart Classification Paradigms for Protein Samples from 1-D Electrophoresis Gel

Electrophoresis allows us to identify the types of proteins present in food, DNA, tissues and more. With the help of the molecular marker their weight is known, these markers are applied within the one-dimensional gel, and their protein value is known by means of marks. In this research, the molecular marker is obtained and the wavelet transform (WT) is obtained, generating approximation coefficients, which were taken to determine a molecular weight using three classification paradigms. The first paradigm is an approach in content-based image retrieval (CBIR) which makes a detection of the molecular weight in electrophoresis samples. The second approach is a neural network, thus two models are employed: self-organization maps (SOM) and back propagation in a supervised and unsupervised way, respectively. The third approach is based in a J48 decision tree. A comparison is made between the three paradigms for molecular weight computation. Neural networks obtained an improvement in the precision compared versus the CBIR-WT. Five parametric statistics were generated from the wavelet approximation coefficients. The CBIR-WT, SOM, back propagation and J48 were decisive for the classification and calculation of the molecular weight of each protein stain in the one-dimensional electrophoresis gel.

Differential levels of Alpha-1-inhibitor III, Immunoglobulin heavy chain variable region, and Hypertrophied skeletal muscle protein GTF3 in rat mammary tumorigenesis

Early detection of breast cancer can be best facilitated by the development of precancerous markers. Serum proteins being the sensitive signatures, can be the ideal choice. We previously demonstrated the reduced levels of two serum proteins at a very early stage of tumorigenesis in a breast cancer model, developed in Wistar rats by 7,12-dimethylbenz[a]anthracene (DMBA) administration. Here we report the dysregulation of three more proteins in the serum collected at another early stage (15 weeks) of tumorigenesis in the same model. The proteins were identified (as Alpha-1-inhibitor III (Mug1), Immunoglobulin heavy chain variable region (IGHV), and Hypertrophied skeletal muscle protein GTF3) by MALDI-TOF MS after the screening and fingerprinting of serum samples by one-dimensional (1D) and two-dimensional (2D) electrophoresis respectively. Relative expression analysis of corresponding genes was also carried out, and the results were found as supporting the proteomic findings. In addition, the candidate proteins of the study and their corresponding ribonucleic acids (RNAs) were subjected to homology modelling and docking (using softwares like MODELLER, 3dRNA, Autodock4.0, and GROMACS etc), which revealed the binding sites for carcinogen (DMBA) and its nature of interaction with proteins and RNAs. Moreover, the network analysis by GeneMANIA unraveled the protein/gene functional network in which Mug1, IGHV, and GTF3 are involved. Based on the significant protein and gene expression alterations in early tumorigenesis, these proteins may prove very effective in search for biomarkers for the early detection of mammary cancer. Further, these proteins can also be tried as targets for chemotherapy.

A Preliminary Study: Troponin T and Reg3β in Children with Left-to-Right Shunt Congenital Heart Disease with Heart Failure

Background: Congenital heart disease (CHD) cause heart failure and myocardial injury. Troponin in the heart is a biomarker of myocardial injury in adults and children. Studies that examine troponin T and Reg3β in children with left-to-right shunt CHD with heart failure are still limited. Objective: This study aims to analyse the troponin T and Reg3β in children with left-to-right shunt CHD with heart failure compared to children without heart failure. Method: This study was a case control study of children with left-to-right shunt CHD with heart failure and children with left-to-right shunt CHD without heart failure performed with non-random sampling consecutive techniques at the Dr. Soetomo General Hospital, Surabaya in April–June 2019. The diagnosis of left-to-right shunt CHD was determined based on echocardiographic examination. All subjects with left-to-right shunt CHD were evaluated using the Paediatric Heart Failure Score. Troponin T examination was carried out using a one-dimensional electrophoresis technique, which is 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Reg3β examination was carried out by the ELISA method. Data analysis was performed with an independent sample t test using the SPSS. Results: This study involved 11 children, consisting of 7 children with left-to-right shunt CHD with heart failure and 4 children with left-to-right shunt CHD without heart failure. Most children (72.7%) were female, 3 children (27.3%) were > 5 years old, 5 children (45.4%) were 5–10 years old and 3 children (27.3%) were > 10 years old. There was a significant increase in the Troponin T and Reg3β in children with left-to-right shunt CHD with heart failure as compared to children without heart failure. Conclusion: Troponin T and Reg3β can be used as biomarkers in children with left-to-right shunt CHD with heart failure.

Comparative proteomic analysis of mature and immature oocytes in domestic cats

Objective: To evaluate changes of feline (Felis catus) oocytes proteins during in vitro maturation by using the proteomic approach. Methods: Immature oocytes (germinal vesicle) isolated from female cats were cultured and collected at 0 h and 24 h. After collection, oocytes were investigated into immature (germinal vesicle) and mature (metaphase II) stages. The qualitative profiles of the proteins at the immature and mature stages were determined by one-dimensional electrophoresis and liquid chromatography-mass spectrometry. Results: Our data revealed that following 24 h in vitro maturation the maturation rate (metaphase II stage) was 58.7%. Eighty-one of the 260 proteins analyzed were differentially expressed between the germinal vesicle stage and the metaphase II -arrest stage. Proteomic analysis of germinal vesicle and metaphase II oocytes showed abundant expression of proteins involved in transportation (10%), indicating that this was a major characteristic of germinal vesicle oocytes. Similarly, analysis of the proteome of metaphase II oocytes indicated that cell cycle proteins were overexpressed. Interestingly, proteins involved in DNA repair and apoptosis were only expressed in germinal vesicle oocytes and proteins involved in fertilization were only expressed in metaphase II oocytes. Conclusions: The overexpression of certain proteins in germinal vesicle and metaphase II is necessary for oocyte development and maturation. Our findings provide a valuable resource for further investigations into protein expression in oocytes at different developmental stages.

Classification of Clinical Isolates of Klebsiella pneumoniae Based on Their in vitro Biofilm Forming Capabilities and Elucidation of the Biofilm Matrix Chemistry With Special Reference to the Protein Content.

Klebsiella pneumoniae is a human pathogen, capable of forming biofilms on abiotic and biotic surfaces. The limitations of the therapeutic options against Klebsiella pneumoniae is actually due to its innate capabilities to form biofilm and harboring determinants of multidrug resistance. We utilized a newer approach for classification of biofilm producing Klebsiella pneumoniae isolates and subsequently we evaluated the chemistry of its slime, more accurately its biofilm. We extracted and determined the amount of polysaccharides and proteins from representative bacterial biofilms. The spatial distribution of sugars and proteins were then investigated in the biofilm matrix using confocal laser scanning microscopy (CLSM). Thereafter, the extracted matrix components were subjected to sophisticated analysis incorporating Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, one-dimensional gel-based electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and MALDI MS/MS analysis. Besides, the quantification of its total proteins, total sugars, uronates, total acetyl content was also done. Results suggest sugars are not the only/major constituent of its biofilms. The proteins were harvested and subjected to SDS-PAGE which revealed various common and unique protein bands. The common band was excised and analyzed by HPLC. MALDI MS/MS results of this common protein band indicated the presence of different proteins within the biofilm. The 55 different proteins were identified including both cytosolic and membrane proteins. About 22 proteins were related to protein synthesis and processing while 15 proteins were identified related to virulence. Similarly, proteins related to energy and metabolism were 8 and those related to capsule and cell wall synthesis were 4. These results will improve our understanding of Klebsiella biofilm composition and will further help us design better strategies for controlling its biofilm such as techniques focused on weakening/targeting certain portions of the slime which is the most common building block of the biofilm matrix.

Assessment of RNAlater® as a Potential Method to Preserve Bovine Muscle Proteins Compared with Dry Ice in a Proteomic Study.

RNAlater® is regarded as a potential preservation method for proteins, while its effect on bovine muscle proteins has rarely been evaluated. Bovine muscle protein samples (n = 12) collected from three tender (Warner–Bratzler shear force: 30.02–31.74 N) and three tough (Warner–Bratzler shear force: 54.12–66.25 N) Longissimus thoracis et lumborum (LTL) samples, preserved using two different sampling preservation methods (RNAlater® and dry ice), at two post mortem time points (day 0 and day 14), were characterized using one-dimensional electrophoresis. Fourteen bands with molecular weights ranging from 15 to 250 kDa were verified, both in the dry ice and RNAlater® storage groups, at each time point, using image analysis. A shift from high to low molecular weight fragments, between day 0 and day 14, indicated proteolysis of the muscle proteins during post mortem storage. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and database searching resulted in the identification of 10 proteins in four bands. Protein profiles of muscle preserved in RNAlater® were similar to those of muscle frozen on dry ice storage, both at day 0 and day 14. The results demonstrate that RNAlater® could be a simple and efficient way to preserve bovine muscle proteins for bovine muscle proteomic studies.

Optimization Method for Proteomic Analysis of the Larva and Adult Tissues of Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Plutella xylostella (L.) (Lepidoptera: Plutellidae), the major insect pest of cruciferous crops worldwide shows significant resistance to almost all classes of insecticides. In order to effectively prevent and manage the insecticidal resistance, it is crucial to understand the physiological adaptation of insects against insecticides. Identification of insect protein that interacting with insecticides and characterization of their modification in resistant strains can be done by using differential proteomics approach. This study focuses on optimizing a sensitive and rapid method for the extraction of high quality protein of both larva and adult tissues of P. xylostella to be used in two-dimensional gel electrophoresis. Five extraction methods were evaluated for protein concentration, yields and resolving patterns of one-dimensional and two-dimensional electrophoresis. The results showed that trichloroacetic acid/acetone extraction methods with two different concentrations of 2-mercaptoethanol produced the highest protein concentration and yield for both adult and larva tissues, respectively. Meanwhile, trichloroacetic acid/acetone with dithiothreitol extraction method gave better separation of spots and intensity for both larva and adult tissues compared to other methods tested. As such, we concluded that trichloroacetic acid/acetone with dithiothreitol successfully yielded high total protein concentration and good separation of two-dimensional electrophoresis gel spots in both adult and larva P. xylostella.

Електрофорез білків крові собак з пухлинами молочної залози

Many publications are devoted to the study of proteins that are associated with the onset and progression of mammary tumors. The researchers study proteins that have a diagnostic value for determining the stage of the tumors and their types. This is necessary for early diagnosis and successful treatment. The aim of this work was to study blood proteins from canine with mammary tumors and to determine the fractions that change by the disease. The proteins were fractionated by a method of one-dimensional electrophoresis in a polyacrylamide gel. Sick animals with breast tumors had the increased content of γ-globulins by 37-88% (Yorkshire Terrier 4 years, Yorkshire Terrier 12 years old and mixed-breed of German Shepherd Dog 8 years old) in comparison with healthy animals (German Shepherd Dogs 2, 5 and 7 years old). Rottweiler with breast tumor (9 years) had 15% of the γ-globulins increase only. These immunoglobulins didn't change ten days after the operation. Only sick mixed-breed German Shepherd Dog had significantly increased levels of haptoglobin. This dog was also observed with the several times increase of the ceruloplasmin concentration. But sick Yorkshire Terriers and healthy animals had similar content of protein. Rottweiler also had similar ceruloplasmin concentration to healthy animals. There was observed significant increase of this fraction (from 0.14 to 5.14). in 10 days after the surgery. The content of transferrin increased (11.72), but after the surgery, it returned to normal (3.4) and didn't differ from healthy animals. Among the all sick animals only Rottweiler had lower content of albumin than healthy animals, and it didn't change 10 days after the surgery. Keywords: dogs, protein, blood, mammary tumor