Two-dimensional Polyacrylamide Gel Electrophoresis Research Articles

Feedlot diets containing different starch levels and additives change the cecal proteome involved in cattle\u2019s energy metabolism and inflammatory response

Diets for feedlot cattle must be a higher energy density, entailing high fermentable carbohydrate content. Feed additives are needed to reduce possible metabolic disorders. This study aimed to analyze the post-rumen effects of different levels of starch (25%, 35%, and 45%) and additives (monensin or a blend of essential oils and exogenous α-amylase) in diets for Nellore feedlot cattle. The cecum tissue proteome was analyzed via two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and then differentially expressed protein spots were identified with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The use of blends of essential oils associated with α-amylase as a feed additive promoted the upregulation of enzymes such as triosephosphate isomerase, phosphoglycerate mutase, alpha-enolase, beta-enolase, fructose-bisphosphate aldolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), l-lactate dehydrogenase B, l-lactate dehydrogenase A chain, l-lactate dehydrogenase, and ATP synthase subunit beta, which promote the degradation of carbohydrates in the glycolysis and gluconeogenesis pathways and oxidative phosphorylation, support pyruvate metabolism through the synthesis of lactate from pyruvate, and participate in the electron transport chain, producing ATP from ADP in the presence of a proton gradient across the membrane. The absence of proteins related to inflammation processes (leukocyte elastase inhibitors) in the cecum tissues of animals fed essential oils and amylase may be because feed enzymes can remain active in the intestine and aid in the digestion of nutrients that escape rumen fermentation; conversely, the effect of monensin is more evident in the rumen and less than 10% results in post-ruminal action, corroborating the hypothesis that ionophore antibiotics have a limited effect on the microbiota and intestinal fermentation of ruminants. However, the increase in starch in these diets promoted a downregulation of enzymes linked to carbohydrate degradation, probably caused by damage to the cecum epithelium due to increased responses linked to inflammatory injuries.

Proteomics Study of Mesenchymal Stem Cell-Like Cells Obtained from Tumor Microenvironment of Patients with Malignant and Benign Salivary Gland Tumors.

Objective Salivary gland tumors (SGTs) show some aggressive and peculiar clinicopathological behaviors that might be related to the components of the tumor microenvironment, especially mesenchymal stem cells (MSCs)-associated proteins. However, the role of MSCs-related proteins in SGTs tumorigenesis is poorly understood. This study aimed to isolate and characterize MSCs from malignant and benign tumor tissues and to identify differentially expressed proteins between these two types of MSCs. Materials and Methods In this experimental study, MSC-like cells derived from benign (pleomorphic adenoma, n=5) and malignant (mucoepidermoid carcinoma, n=5) tumor tissues were verified by fluorochrome antibodies and flow cytometric analysis. Differentially expressed proteins were identified using two-dimensional polyacrylamide gel electrophoresis (2DE) and Mass spectrometry. Results Results showed that isolated cells strongly expressed characteristic MSCs markers such as CD44, CD73, CD90, CD105, and CD166, but they did not express or weakly expressed CD14, CD34, CD45 markers. Furthermore, the expression of CD24 and CD133 was absent or near absent in both isolated cells. Results also discovered overexpression of Annexin A4 (Anxa4), elongation factor 1-delta (EF1-D), FK506 binding protein 9 (FKBP9), cytosolic platelet-activating factor acetylhydrolase type IB subunit beta (PAFAH1B), type II transglutaminase (TG2), and s-formylglutathione hydrolase (FGH) in MSCs isolated from the malignant tissues. Additionally, heat shock protein 70 (Hsp70), as well as keratin, type II cytoskeletal 7 (CK-7), were found to be overexpressed in MSCs derived from the benign ones. ConclusionMalignant and benign SGTs probably exhibit a distinct pattern of tissue proteins that are most likely related to the metabolic pathway. However, further studies in a large number of patients are required to determine the applicability of identified proteins as new targets for cancer therapy.

Importance of Proteomics in Bioremediation

Abstract: Bioremediation can be used to help clean up contaminated regions today since environmental contamination is a major issue. The use of microbial mediated bioremediation offers a lot of potential for effectively restoring a polluted environment, but a lack of detail about the parameters that determine whether certain microbial communities can grow and reproduce in a polluted environment makes this kind of bioremediation challenging. Transcriptomics, proteomics, and interactomics are flourishing fields that hold great potential for addressing long-standing issues about the molecular mechanisms that drive the mineralization pathway. With the aid of microarray technology, transcriptomic approaches have been applied to study the structure and expression of transcripts during mineralisation. However, transcripts cannot generally cause any physiological effect; instead, they must be translated into proteins with significant functions. Two-dimensional polyacrylamide gel electrophoresis (2-DE) is a powerful tool for identifying these proteins via proteomic techniques. The current advancements in mass spectrometry (MS) and protein microarrays are playing an important role in functional proteomics. A comprehensive genome-wide analysis of differentially expressed proteins and a screen for proteins that interact with specific mineralization factors would allow us to gain a better understanding of bioremediation. Keywords: bioremediation, transcriptomics, proteomics, interactomics, microarray

Synovial fluid proteome profile of surgical versus chemical induced osteoarthritis in rabbits.

BackgroundAnimal models are significant for understanding human osteoarthritis (OA). This study compared the synovial fluid proteomics changes in surgical and chemical induced OA models.MethodsThirty rabbits either had anterior cruciate ligament transection (ACLT) procedure or injected intra-articularly with monosodium iodoacetate (MIA, 8 mg) into the right knee. The joints were anatomically assessed, and the synovial fluid proteins analyzed using two-dimensional polyacrylamide gel electrophoresis (2DGE) and MALDI TOF/TOF mass spectrometry analysis at 4, 8 and 12 weeks. The proteins’ upregulation and downregulation were compared with control healthy knees.ResultsSeven proteins (histidine-rich glycoprotein, beta-actin-like protein 2 isoform X1, retinol-binding protein-4, alpha-1-antiproteinase, gelsolin isoform, serotransferrin, immunoglobulin kappa-b4 chain-C-region) were significantly expressed by the surgical induction. They characterized cellular process (27%), organization of cellular components or biogenesis (27%), localization (27%) and biological regulation (18%), which related to synovitis, increased cellularity, and subsequently cartilage damage. Three proteins (apolipoprotein I-IV precursor, serpin peptidase inhibitor and haptoglobin precursor) were significantly modified by the chemical induction. They characterized stimulus responses (23%), immune responses (15%), biological regulations (15%), metabolism (15%), organization of cellular components or biogenesis (8%), cellular process (8%), biological adhesions (8%) and localization (8%), which related to chondrocytes glycolysis/death, neovascularization, subchondral bone necrosis/collapse and inflammation.ConclusionsThe surgical induced OA model showed a wider range of protein changes, which were most upregulated at week 12. The biological process proteins expressions showed the chemical induced joints had slower OA progression compared to surgical induced joints. The chemical induced OA joints showed early inflammatory changes, which later decreased.

Purification and Identification of the 20S Proteasome Complex from Zebrafish.

The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish (Danio rerio) from the whole body. The cytosolic fraction of zebrafish hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA), a well-known substrate for the proteasome, in the presence of sodium dodecyl sulfate. From the cytosolic fraction, the 20S proteasome was purified using five column chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S-300 gel, hydroxylapatite, and phenyl Sepharose. Electrophoresis and Western blot analyses showed that zebrafish 20S proteasome subunits have molecular masses ranging from 22 to 33 kDa. The subunit composition of the purified 20S proteasome was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation. Fourteen kinds of 20S subunits were found. As a special characteristic of zebrafish, two proteins of the α1 subunit were identified. In addition, the results suggested that the α8 subunit is in the 20S complex instead of the α4 subunit. In this study, we demonstrated the subunit composition of the 20S proteasome complex present in zebrafish cells.

MatGel: A MATLAB program for quantitative analysis of 2D polyacrylamide electrophoresis (2D-PAGE) protein gel images

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is widely used in proteomics studies. Hundreds of proteins extracted from a biological sample can be separated and visualized on a 2D-PAGE gel. The interpretation of protein expression levels relies on the comparison of areas and intensities of the corresponding protein spots in 2D-PAGE gel images. However, determination of protein spot areas by the manual selection method is time-consuming and error-prone. The purpose of this research is to develop a highly automated program for the simultaneous detection and quantification of protein spots across a large number of 2D-PAGE protein gel images by using MATLAB image processing toolbox. This program will enhance the potential of using 2D-PAGE technique as a high throughput quantitative protein expression study tool. We developed MatGel, a simple and efficient program for protein spot area detection and intensity quantification from 2D-PAGE protein gel images. MatGel can detect and output the areas and mean intensities of corresponding protein spots across a large number of 2D-PAGE gel images simultaneously. Users also have options to adjust preferences at each step of image analysis. Basic knowledge with MATLAB programming language is required to run the program.•We developed MATLAB program MatGel to automate the determination of protein spots on 2D-PAGE protein gel images.•MatGel can analyze a large number of 2D-PAGE gel images simultaneously to minimize human errors.•MatGel is flexible and easy to use.

Two-Dimensional Polyacrylamide Gel Electrophoresis Analysis of Viroid RNAs.

The circular and linear forms of viroid RNAs can be separated by two-dimensional polyacrylamide gel electrophoresis (PAGE) based on the selective delay in mobility that circular RNAs experience under denaturing conditions. First PAGE separates RNA preparations from viroid-infected plants, and the whole lane from this first gel is next perpendicularly loaded on top of a second gel. Separation continues under new conditions that differ in the degree of denaturation from the first. The result is a two-dimensional separation of the RNAs in which circular and linear molecules are distributed in two parallel diagonals.

Proteomic Analysis of Common Bean (Phaseolus vulgaris L.) by Two-Dimensional Gel Electrophoresis and Mass Spectrometry

The modern cultivated common bean (Phaseolus vulgaris) has evolved from wild common beans distributed in Central America, Mexico and the Andean region of South America. It has been reported that wild common bean accessions have higher levels of protein content than the domesticated dry bean cultivars. However, there is limited proteomic analysis of wild or domesticated common beans. In this investigation, we reported a proteomic analysis of a wild bean from Mexico. We utilized the TCA/Acetone method for protein extraction, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation, and subsequent mass spectrometry (MS) for protein identification. Proteins were separated in the first dimension using pH range from 4-7. A total of 237 protein spots from the second dimension were isolated, digested with trypsin, and analyzed by MALDI/TOF/TOF mass spectrometry. We identified 141 protein spots by searching NCBI non redundant databases using the Mascot search engine and found a total of 43 unique proteins. Gene Ontology (GO) analysis was employed to understand the molecular processes in which the identified common bean proteins are involved. The majority of proteins are involved in binding (41.5%) and catalytic activity (35.8%), followed by nutrient reservoir activity (7.5%), antioxidant activity (1.9%), transporter activity (3.8%), enzyme regulator activity (3.8%), structural molecule activity (1.9%), and electron carrier activity (3.8%). The results indicate that TCA/Acetone extraction and 2D-PAGE is efficient in separating common bean proteins. Further identification of these proteins by MS demonstrates the quality of this protein extraction method. These results will be useful for the development of value added common beans by alteration of protein components.

Analysis of Soybean Embryonic Axis Proteins by Two-Dimensional Gel Electrophoresis and Mass Spectrometry

A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and subsequent mass spectrometry (MS) for protein identification was applied to establish a proteomic reference map for the soybean embryonic axis. Proteins were extracted from dissected embryonic axes and separated in the first dimension using a pH range from 4-7. A total of 401 protein spots were isolated, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 335 protein spots by searching National Center for Biotechnology Information (NCBI) non redundant databases using the Mascot search engine and found a total of 200 unique proteins. Gene Ontology (GO) analysis was employed to understand the molecular processes in which the identified embryonic axes proteins are involved. The majority of proteins play a functional role in catalytic activity (42.9%) and binding (39.3%), followed by nutrient reservoir activity (5.3%), structural molecular activity (4.0%), antioxidant activity (3.2%), transporter activity (2.4%), enzyme regulator activity (1.2%), molecular transducer activity (0.8%), and transcription regulator activity (0.8%). Our 2D-profiling of soybean axis proteins has established for the first time a baseline proteome on which to investigate and compare factors affecting soybean embryonic development and the interaction of beneficial and pathogenic soilborne organisms during seed germination.

Skeletal muscle atrophy-induced hemopexin accelerates onset of cognitive impairment in Alzheimer's disease.

BackgroundAlzheimer's disease (AD) is an unmet medical need worldwide, and physical inactivity is a risk factor for AD. Performing physical exercise is difficult at old age, and thus, decline in physical movement may be a cause of age‐associated lowering of the brain function. This study aimed to elucidate the molecular mechanism and onset of the skeletal muscle atrophy‐induced acceleration of AD.MethodsPre‐symptomatic young 5XFAD or non‐transgenic wildtype mice were used. The bilateral hindlimbs were immobilized by placing them in casts for 14 days. Cognitive function was evaluated using the object recognition and spatial memory tests. Further, the hindlimb muscles were isolated for organ culture. Conditioned media (CM) of each muscle was separated by two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). Protein expressions in the CM were analysed by matrix‐assisted laser desorption/ionization‐time‐of‐flight mass spectrometry analysis. The expression levels of candidate proteins were quantified using ELISA. After continuous intracerebroventricular (i.c.v.) infusion of recombinant hemopexin, cognitive function was evaluated. Gene microarray analysis of the hippocampus was performed to investigate the molecules involved in the accelerated memory deficit. Real‐time reverse transcription polymerase chain reaction and histological analysis confirmed the expression.ResultsCasting for 2 weeks reduced skeletal muscle weight. Object recognition memory in the cast‐attached 5XFAD mice (n = 7, training vs. test, P = 0.3390) was impaired than that in age‐matched wildtype (n = 7, training vs. test, P = 0.0523) and non‐cast 5XFAD mice (n = 7, training vs. test, P = 0.0473). On 2D‐PAGE, 88 spots were differentially expressed in muscle CM. The most increased spot in the cast‐attached 5XFAD CM was hemopexin. Hemopexin levels in the skeletal muscle (n = 3, P = 0.0064), plasma (n = 3, P = 0.0386), and hippocampus (n = 3, P = 0.0164) were increased in cast‐attached 5XFAD mice than those in non‐cast 5XFAD mice. Continuous i.c.v. infusion of hemopexin for 2 weeks induced memory deficits in young 5XFAD mice (n = 4, training vs. test, P = 0.6764). Lipocalin‐2 (Lcn2) messenger RNA (mRNA), neuroinflammation‐associated factor, was increased in the hippocampus in hemopexin‐infused 5XFAD mice than in control mice. LCN2 protein in the hippocampus was localized in the neurons, but not glial cells. Lcn2 mRNA levels in the hippocampus were also increased by cast‐immobilization of the hindlimbs (n = 6, P = 0.0043).ConclusionsThese findings provide new evidence indicating that skeletal muscle atrophy has an unbeneficial impact on the occurrence of memory impairment in young 5XFAD mice, which is mediated by the muscle secreted hemopexin.

Molecular Profiles of Amyloid-\u03b2 Proteoforms in Typical and Rapidly Progressive Alzheimer\u2019s Disease

The molecular determinants of atypical clinical variants of Alzheimer’s disease, including the recently discovered rapidly progressive Alzheimer’s disease (rpAD), are unknown to date. Fibrilization of the amyloid-β (Aβ) peptide is the most frequently studied candidate in this context. The Aβ peptide can exist as multiple proteoforms that vary in their post-translational processing, amyloidogenesis, and toxicity. The current study was designed to identify these variations in Alzheimer’s disease patients exhibiting classical (sAD) and rapid progression, with the primary aim of establishing if these variants may constitute strains that underlie the phenotypic variability of Alzheimer’s disease. We employed two-dimensional polyacrylamide gel electrophoresis and MALDI-ToF mass spectrometry to validate and identify the Aβ proteoforms extracted from targeted brain tissues. The biophysical analysis was conducted using RT-QuIC assay, confocal microscopy, and atomic force microscopy. Interactome analysis was performed by co-immunoprecipitation. We present a signature of 33 distinct pathophysiological proteoforms, including the commonly targeted Aβ40, Aβ42, Aβ4-42, Aβ11-42, and provide insight into their synthesis and quantities. Furthermore, we have validated the presence of highly hydrophobic Aβ seeds in rpAD brains that seeded reactions at a slower pace in comparison to typical Alzheimer’s disease. In vitro and in vivo analyses also verified variations in the molecular pathways modulated by brain-derived Aβ. These variations in the presence, synthesis, folding, and interactions of Aβ among sAD and rpAD brains constitute important points of intervention. Further validation of reported targets and mechanisms will aid in the diagnosis of and therapy for Alzheimer’s disease.

Effect of Heat Treatment on the Property, Structure, and Aggregation of Skim Milk Proteins.

To study the mechanism of heat-induced protein aggregates, skim milk was heated at 55, 65, 75, 85, and 95°C for 30 s. Then, the sulfhydryl content, surface hydrophobicity, and secondary structure of heat-treated skim milk were studied. Treating skim milk at different temperatures induced a decrease in sulfhydryl content (75.9% at 95°C) and an increase in surface hydrophobicity (44% at 95°C) with a disrupted secondary structure containing random coil, β-sheet, and β-turn of skim milk proteins. The change in these properties facilitated aggregate formation through disulfide bonds and hydrophobicity interaction. Microstructural observation also showed a higher degree of aggregation when skim milk was heated at 85 and 95°C. The result of two-dimensional polyacrylamide gel electrophoresis demonstrated that the aggregates consisted of a high proportion of κ-casein, β-lactoglobulin, and other whey proteins.

Investigation of Protein Biomarkers and Oxidative Stress in Pinirampus pirinampu Exposed to Mercury Species from the Madeira River, Amazon-Brazil.

In recent decades, the scientific community has widely debated the contamination of fish in the Amazon region by mercury species. As the diet of riverside populations in the Amazon region is based mainly on fish, these populations are exposed to mercurial species that can cause serious and irreversible damage to their health. The risks of consuming fish exposed to mercurial species in the Amazon region have motivated toxicological investigations. However, the effect of mercurial species on protein and enzyme levels is still controversial. In this work, analytical and bioanalytical techniques Two-dimensional polyacrylamide gel electrophoresis [2D-PAGE] Graphite Furnace Atomic Absorption Spectrometry [GFAAS], and Mass Spectrometry in Sequence with Electrospray Ionization [ESI-MS/MS] were used to identify proteins associated with mercury (metal-binding protein) in muscle and liver tissues of the fish species Pinirampus pirinampu from the Madeira River, in the Brazilian Amazon. Enzymatic and lipid peroxidation analyses were also used to assess changes related to oxidative stress. Determinations of total mercury by GFAAS indicated higher concentrations in liver tissue (555 ± 19.0µgkg-1) when compared to muscle tissue (60 ± 2.0µgkg-1). The fractionation process of tissue proteomes by 2D-PAGE and subsequent mapping of mercury by GFAAS in the protein spots of the gels identified the presence of mercury in three spots of the liver tissue (concentrations in the range of 0.800 to 1.90mgkg-1). The characterization of protein spots associated with mercury by ESI-MS/MS identified the enzymes triosephosphate isomerase A, adenylate kinase 2 mitochondrial, and glyceraldehyde-3-phosphate dehydrogenase as possible candidates for mercury exposure biomarkers. The muscle tissue did not show protein spots associated with mercury. Enzymatic activity decreased proportionally to the increase in mercury concentrations in the tissues.

ABIOTIC STRESS RESPONSE-ASSOCIATED PROTEINS IN A SALT TOLERANT STRAIN OF THE CYANOBACTERIUM FREMYELLA DIPLOSIPHON: A POTENTIAL BIOFUEL AGENT

Cyanobacteria have gained great significance as a clean green alternative to fossil fuels as they are renewable and sustainable. Fremyella diplosiphon is a potential biofuel-producing cyanobacterium that efficiently captures light energy for photosynthesis. Efforts in our laboratory have been aimed to identify salt response pathways in this organism to develop strategies to enhance halotolerance in this model organism. In this study, protein expression in wild type and halotolerant mutant F. diplosiphon strains was compared using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time of flight mass spectrometry. Of the 16 up-regulated proteins identified in the mutant, tripartite ATP-independent periplasmic (TRAP) transporter solute receptor was found to assist in salt-stress response with a significant hit to a corresponding spot with a score of 669 and 21% sequence coverage. Another protein, elongation factor Tu, linked to enhanced abiotic stress tolerance was significantly matched to a spot with a score of 1358 and 52% sequence coverage. Results of our study indicate that TRAP transporter solute receptor likely assists in enhancing halotolerance of the mutant, while elongation factor Tu is up-regulated in response to heat shock. These findings enable better understanding of F. diplosiphon salt-stress response paving the way for novel approaches in enhancing its halotolerance, leading to viable options leading to biofuel production.

Characterization of Functional Components in Bovine Colostrum That Inhibit Norovirus Capsid Protruding Domains Interacting with HBGA Ligands.

Human noroviruses (huNoVs) cause epidemic acute gastroenteritis with significant mortality and morbidity worldwide. However, there are no commercial vaccines or antivirals against these important pathogens so far. In this study, we found that bovine colostrum (bCM) inhibited huNoV VLPs and their capsid-protruding (P) domains binding to histo-blood group antigens (HBGAs) that are huNoV receptor or attachment factors for infection, suggesting that bCM may function as a natural antiviral against huNoVs. We then characterized the bCM for the functional inhibition components by sequentially separating bCM into multiple fractions through various chromatography approaches, followed by determining their inhibitory abilities against huNoV receptor-binding P protein interacting with HBGAs. The protein components of bCM functional fractions were examined by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Our data suggested that some milk proteins, likely in the form of glycoproteins, contribute to the observed blocking effects of bCM. Our findings lay an important foundation to further develop bCM into a potential natural antiviral against huNoVs.

Gene Expression and Proteomics Studies Suggest an Involvement of Multiple Pathways Under Day and Day-Night Combined Heat Stresses During Grain Filling in Wheat.

Recent weather fluctuations imposing heat stress at the time of wheat grain filling cause frequent losses in grain yield and quality. Field-based studies for understanding the effect of terminal heat stress on wheat are complicated by the effect of multiple confounding variables. In the present study, the effect of day and day–night combined heat stresses during the grain-filling stage was studied using gene expression and proteomics approaches. The gene expression analysis was performed by using real-time quantitative PCR (RT-qPCR). The expression of genes related to the starch biosynthetic pathway, starch transporters, transcription factors, and stress-responsive and storage proteins, at four different grain developmental stages, indicated the involvement of multiple pathways. Under the controlled conditions, their expression was observed until 28 days after anthesis (DAA). However, under the day stress and day–night stress, the expression of genes was initiated earlier and was observed until 14 DAA and 7 DAA, respectively. The protein profiles generated using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS/MS) showed a differential expression of the proteins belonging to multiple pathways that included the upregulation of proteins related to the translation, gliadins, and low-molecular-weight (LMW) glutenins and the downregulation of proteins related to the glycolysis, photosynthesis, defense, and high-molecular-weight (HMW) glutenins. Overall, the defense response to the day heat stress caused early gene expression and day–night heat stress caused suppression of gene expression by activating multiple pathways, which ultimately led to the reduction in grain-filling duration, grain weight, yield, and processing quality.

Comparative Proteomics Analysis of Phosphine-Resistant and Phosphine-Susceptible Sitophilus oryzae (Coleoptera: Curculionidae)

A proteomic method combining two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization-time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF) was used to compare the protein expression profiles of phosphine-resistant (PH3-R) and -susceptible (PH3-S) strains of Sitophilus oryzae. Thirty-nine differentially expressed protein spots were identified between the PH3-R and PH3-S strains; 20 protein spots were upregulated, and 19 protein spots were downregulated in the PH3-R strain compared with their expression in the PH3-S strain. In particular, cytochrome oxidase subunit I showed 15-fold higher expression in the PH3-R strain than in the PH3-S strain. Additionally, citrate synthase 2, delta-1-pyrolline-5-carboxylate dehydrogenase, and triose-phosphate isomerase were highly expressed in the PH3-R strain. In summary, our study has improved understanding of the molecular mechanisms of phosphine resistance in the rice weevil.

Study on the Correlation between the Protein Profile of Lupin Milk and Its Cheese Production Compared with Cow's Milk.

Australian sweet lupin, the largest legume crop grown in Western Australia, is receiving global attention from the producers of new foods. To understand the effect of protein on cheese yield, lupin milk proteins were separated from the first, second, and third filtrations by cheesecloths. However, proteins from the first and second were analyzed using two-dimensional polyacrylamide gel electrophoresis; then, the isolated proteins associated with cheese production were identified. The research also focused on identifying the optimal method of cheese production based on the coagulation process, temperature, yield, and sensory evaluation. Lupin curds from the two cultivars, Mandelup and PBA Jurien, were produced using vinegar, lemon juice, starter culture, vegetable rennet enzyme as coagulant, as well as curd generated using starter culture and vegetable rennet enzyme. Cow’s milk was used as a control. The results indicated that first-time filtration produced better extraction and higher yield of lupin proteins and cheese than the second filtration. A sensory analysis indicated that lupin cheese produced from PBA Jurien lupin milk using vinegar, 7.80% expressed as acetic acid, and ground in 45 °C water, was the most acceptable. The cheeses were examined for their protein, carbohydrates, fat, ash, and moisture contents. The concentration of protein was approximately 27.3% and 20.6%, respectively, in the cheese from PBA Jurien and Mandelup. These results suggest that lupin milk can adequately supply the proteins needed in human diets and, thus, could be used in the production of many existing products that require animal milk as an input.

Heating Effect on Protein’s Aggregation in Goat and Bovine Milk Based on One and Two-Dimensional Polyacrylamide Gel Electrophoresis

Goat milk has been considered as a possible alternative of bovine milk because of hypoallergenic. The milk protein is the major allergenic substances, however, the differences of proteins have been few demonstrated in processing between goat and bovine milk. To explore the influence of three heat treatment and ultrahigh pressure (UHP, at 500 MPa and 17 °C) on milk, we investigated the differences of proteins in the two types of milk by one and two-dimensional polyacrylamide gel electrophoresis (1D and 2D PAGE) methods. β-lactoglobulin (β-LG) from goat milk showed one big band and migrated slower than α-lactalbumin (α-LA) on the native gel; while bovine milk showed two β-LG bands that had higher mobility ratio than other protein bands. The pasteurizing (63 °C, 30 min) and UHT (143 °C, 15 s) led to no obvious missing of protein bands, but the boiling treatment (100 °C, 3 min) caused binding of whey protein with κ-CN and αs2-CN in both types of milk. UHP treatment of milk also causes β-LG aggregates with casein by forming disulfide-bonded. All of the treatments indicated that goat milk was more sensitive to heat and pressure than bovine milk to form the aggregates and heat processing had the greatest effect on β-LG. This study could be helpful in understanding the difference in technological-functional properties of the two types of milk.

Identification of Bacterial Proteins in the Gut of Sunn pest, Eurygaster integriceps

Proteomics is a powerful tool to identify effective proteins in the biochemical reactions of the insect body. Many proteins were reported in the gut lumen and tissues which are essential to complete the physiological role of the alimentary canal of Sunn pest. The gut microbiome of insects has a key role in the digestive process. In this study, for the first time, gut proteins of adult Sunn pest were extracted. These proteins were visualized and identified with two-dimensional polyacrylamide gel electrophoresis and mass spectrometry, respectively. Newly identified proteins include pyruvate dehydrogenase, oxidoreductase FAD-binding protein, hypothetical proteins, glycerol-3 phosphate dehydrogenase, conserved hypothetical proteins, ABC excinucleases, ABC-type transport systems and molecular chaperones. The accumulation of these metabolism proteins in the gut of Sunn pest indicates the importance of symbiotic proteins in the improvement of digestive activities and insect-bacteria interactions. Results suggest that the identified bacterial proteins can be considered as effective proteins in the process of nutrition and provide more gut-derived targets for enzyme engineering and development of biopesticides.