Protein Spots Research Articles

Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Str\xe4ussler-Scheinker Syndrome In Vivo and In Vitro

Alteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

Prediction of hot spots in protein\u2013DNA binding interfaces based on supervised isometric feature mapping and extreme gradient boosting

BackgroundIdentification of hot spots in protein-DNA interfaces provides crucial information for the research on protein-DNA interaction and drug design. As experimental methods for determining hot spots are time-consuming, labor-intensive and expensive, there is a need for developing reliable computational method to predict hot spots on a large scale.ResultsHere, we proposed a new method named sxPDH based on supervised isometric feature mapping (S-ISOMAP) and extreme gradient boosting (XGBoost) to predict hot spots in protein-DNA complexes. We obtained 114 features from a combination of the protein sequence, structure, network and solvent accessible information, and systematically assessed various feature selection methods and feature dimensionality reduction methods based on manifold learning. The results show that the S-ISOMAP method is superior to other feature selection or manifold learning methods. XGBoost was then used to develop hot spots prediction model sxPDH based on the three dimensionality-reduced features obtained from S-ISOMAP.ConclusionOur method sxPDH boosts prediction performance using S-ISOMAP and XGBoost. The AUC of the model is 0.773, and the F1 score is 0.713. Experimental results on benchmark dataset indicate that sxPDH can achieve generally better performance in predicting hot spots compared to the state-of-the-art methods.

Expression of cytosolic and thiolated proteome of Musca domestica larvae under oxidative challenge.

The study was aimed to investigate the expression of cytosolic and thiolated proteins of Musca domestica larvae under oxidative stress. Proteins from acute treatment of hydrogen peroxide (LC50 = 21.52% (v/v)) on 3rd stage larvae of housefly were extracted and purified using an activated Thiol Sepharose® for thiolated protein purification. Two dimensional gel electrophoresis was used for visualizing and analyzing expression of cytosolic and thiolated proteins. Protein spots with more than 5 fold of expression change were identified using liquid chromatography- tandem mass spectrometry (LC-MS/MS). The cytosolic proteins were actin, tropomyosin, ubiquitin, arginine kinase, pheromone binding protein/general odorant binding protein, and ATP: guanidino phosphotransferase. The thiolated proteins with more than 5 fold change in expression as an effect to the acute treatment were fructose bisphosphate aldolase, short chain dehydrogenase and lactate/malate dehydrogenase. The proteins identified in the study should provide vital information for future reference in oxidative stress defence and response occurring in houseflies.

An Increase in Liver Polyamine Concentration Contributes to the Tryptophan-Induced Acute Stimulation of Rat Hepatic Protein Synthesis.

Tryptophan has a unique role as a nutritional signaling molecule that regulates protein synthesis in mouse and rat liver. However, the mechanism underlying the stimulating actions of tryptophan on hepatic protein synthesis remains unclear. Proteomic and metabolomic analyses were performed to identify candidate proteins and metabolites likely to play a role in the stimulation of protein synthesis by tryptophan. Overnight-fasted rats were orally administered L-tryptophan and then sacrificed 1 or 3 h after administration. Four differentially expressed protein spots were detected in rat liver at 3 h after tryptophan administration, of which one was identified as an ornithine aminotransferase (OAT) precursor. OAT is the main catabolic enzyme for ornithine, and its expression was significantly decreased by tryptophan administration. The concentration of ornithine was increased in the liver at 3 h after tryptophan administration. Ornithine is a precursor for polyamine biosynthesis. Significantly increased concentrations of polyamines were found in the liver at 3 h after administration of tryptophan. Additionally, enhanced hepatic protein synthesis was demonstrated by oral administration of putrescine. We speculate that the increase in ornithine level through suppression of OAT expression by tryptophan administration may lead to accelerated polyamine synthesis, thereby promoting protein synthesis in the liver.

Two dimensional proteomic analysis of serum shows immunological proteins exclusively expressed in sulfur mustard exposed patients with long term pulmonary complications

BackgroundDespite more than 30 years after utilization of sulfur mustard or bis (2-chloroethyl) sulfide (SM) by Iraqi troops against Iranian military members and civilians, there are a lot of reported delayed complications for the exposed people. Nonetheless, the molecular mechanism of action from this chemical warfare agent is not recognized yet. Material and methodIn this study, we employed two dimensional gel electrophoresis (2DE) technique to investigate the serum proteins from chemical exposed people compared to non-exposed individuals to provide an inside into molecular mechanism of this chemical agent. Each group was divided into two subgroups including individuals with, and without respiratory complications. For each group, 10 individuals were included after informed consent. ResultThe results showed protein spots, which were exclusively/mainly expressed in chemical exposed patients with complications, including T cell receptor alpha, and hematopoietic cell signal transducer. Also there were protein spots that were expressed only in all exposed groups (with and without complications). On the other hand, we could identify protein spots that were exclusively expressed/altered only in non-exposed group with complications including Pre T-cell antigen receptor, CD40 ligand, and multidrug and toxin extrusion proteins. ConclusionOur investigation could result in identification of proteins that are associated to chemical exposure, as well as those specific for respiratory complications irrespective of chemical exposure. These candidate proteins can be used as biomarker, as well as a base for understanding the molecular mechanism of this chemical agent.

Molecular cloning and expression analysis of BoRAN3 in Brassica oleracea

ABSTRACT According to structural and functional similarities, small GTP-binding proteins can be divided into five distinct families, in which, Ras-related nuclear protein (Ran) GTPases regulates proteins and RNA across nuclear envelope. To excavate differentially expressed proteins in the pistil after pollination, proteomics was recruited. One protein spot was identified as BoRAN3, was down-regulated expressed in the pistil. Phylogenetic tree assay found RAN3 in Brassica oleracea, Eutrema salsugineum, Brassica napus and Brassica rapa grouped into one clade. Compared BoRAN3 expression levels in reproductive organs found it was expressed in all five organs, specifically highly in the pistil. qRT-PCR assays found that BoRAN3 was up-regulated expsed in the early pollination process. Sub-cellular assay demonstrated that BoRAN3 located in the nucleus. GUS assay found that BoRAN3 was expressed in the cotyledon, root, stoma, lateral root primordia, petal, filigree and both the front and the bottom of pistil. Taken together, our results demonstrated that BoRAN3 was highly expressed in the pistil and up-regulated expressed in the pistil during early pollination process. Based on those results, we speculated that the up-regulated expressed of BoRAN3 in the pistil during early pollination process hints that it may related to pollen germination and pollen tube development.

Influenza virus natural sequence heterogeneity in segment 8 affects interactions with cellular RNA-binding proteins and splicing efficiency

Segment 8 mRNAs of influenza virus A/Brevig Misson/1918/1 (H1N1) are poorly spliced compared to segment 8 mRNAs of influenza virus A/Netherlands/178/95 (H3N2). Using oligonucleotide-mediated protein pull down with oligos spanning the entire length of segment 8 of either influenza virus H1N1 or influenza virus H3N2 we identified cellular RNA binding proteins that interacted with oligonucleotides derived from either H1N1 or H3N2 sequences. When the identified hot spots for RNA binding proteins in H1N1 segment 8 mRNAs were replaced by H3N2 sequences, splicing efficiency increased significantly. Replacing as few as three nucleotides of the H1N1 mRNA with sequences from H3N2 mRNA, enhanced splicing of the H1N1 mRNAs. Cellular proteins U2AF65 and HuR interacted preferentially with the 3′-splice site of H3N2 and overexpression of HuR reduced the levels of unspliced H1N1 mRNAs, suggesting that U2AF65 and HuR contribute to control of influenza virus mRNA splicing.

Dynamic proteome changes of wheat developing grains in response to water deficit and high-nitrogen fertilizer conditions

This study investigated grain proteomic profiles in response to water deficit, high nitrogen (N) fertilizer, and their combined treatments in elite Chinese bread wheat cultivar Jingdong 17, using a two-dimensional difference gel electrophoresis (2D-DIGE)-based approach. Water deficit negatively affected the main agronomic traits of wheat and grain yield, while high-N fertilizer had the opposite effects. The application of a high-N fertilizer under water deficit conditions moderately improved kernel development and grain yield. 2D-DIGE led to the identification of 124 differentially accumulated protein (DAP) spots during five different grain developmental stages, corresponding to 97 unique proteins. The more significant changes of DAPs occurred at 10–20 days after flowering. DAPs were involved in carbohydrate metabolism, protein turnover, protein folding, cell cycle control, stress response, nitrogen metabolism, photosynthesis, and energy metabolism. In particular, water deficit caused a significant downregulation of proteins involved in starch biosynthesis, whereas high-N fertilizer led to a significant upregulation of proteins involved in nitrogen metabolism, carbohydrate metabolism, and starch biosynthesis. The combined treatment resulted in a moderate upregulation of DAPs related to carbohydrate metabolism, starch biosynthesis, and nitrogen metabolism. Our results indicated that high-N fertilization could alleviate yield loss caused by water deficit by promoting the accumulation of proteins involved in nitrogen and carbohydrate metabolism.

Abstract 5131: Differentially expressed proteins in esophageal squamous cell carcinoma tissue

Abstract Purpose: To identify differentially expressed proteins among the esophageal squamous cell carcinoma(ESCC) carcinoma tissue, adjacent normal tissue, normal esophageal mucosa, in order to look out the protein related to different stages and study the molecular mechanism and occurrence of the esophageal squamous cell carcinoma. Methods: Tissue specimens of ESCC and the corresponding clinical data database of ESCC patients were established, a total of 69 patients were selected according to distribution of their sex and age. Two-dimensional gel electrophoresis technology to electrophoresis and get the separation protein of cancerous tissues, adjacent tissues and normal esophageal tissues,then ImageMaster 2D Platinum software was used to search, quantify, match and statistical analysise the proteins on the gel image. 4700 Proteomics Analyzer (TOF/TOFTM) was used to analyze protein spots, in which differentially expressed proteins meet standards were further searched in the Matrix Science, London, UK protein database. Results: Totle 44 and 29 kinds of differentially expressed proteins were screened from esophageal cancer tissues, adjacent noncancerous tissues and normal tissues, among which, there were 13 differentially expressed proteins between esophageal cancer tissues and adjacent noncancerous tissues, 14 differentially expressed proteins between esophageal cancer tissues and normal tissues, 17 between esophageal adjacent noncancerous tissues and normal tissues. These differential expression proteins mainly involved in PKC-catalyzed phosphorylation of inhibitory phosphoprotein of myosin phosphatase, nuclear factor of activated T cells(NFAT) pathway, Integrin Signaling Pathway, Cell to Cell Adhesion Signaling, Calmodulin(CaM) nuclear factor of activated T cells, Mineral absorption of calcium binding protein path way(S100), Path way of vascular smooth muscle contraction affe cted by caldesmon, Apoptotic Signaling in Response to DNA Damage path way, which had the function of cytoskeleton structure, protein phosphorylation, acetylation of aminoend, regulation of calcium combination,tumor cell movement, signal transduction, cell differentiation, cell apoptosis, muscle contraction, translation regulation, gene expression, energy metabolism, cell proliferation. Conclusion: This study study achieved differentially expressed protein in ESCC tissue patients, provides new basis for the study of esophageal cancer carcinogenesis mechanism. Citation Format: Fuchun Si. Differentially expressed proteins in esophageal squamous cell carcinoma tissue [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5131.

Protein Expression Analysis of an In Vitro Murine Model of Prostate Cancer Progression: Towards Identification of High-Potential Therapeutic Targets.

Background: Prostate cancer (PC) is the most frequently diagnosed cancer among men worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We interrogated proteomic signatures that embody the transition of PC from an androgen-dependent (AD) to an androgen-independent (AI) state. Methods: We have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC at phenotypic and subcellular levels. We statistically surveyed global protein expression profiles in these cell lines. Differential profiles were functionally interrogated by pathways and protein–protein interaction network analyses. Results: Protein expression pattern analysis revealed a total of 683 proteins, among which 99 were significantly differentially altered in PLum-AI cells as compared to PLum-AD cells (45 increased and 54 decreased). Principal component analysis (PCA) revealed that the two different cell lines clearly separated apart, indicating a significant proteome expression difference between them. Four of the proteins (vimentin, catalase, EpCAM, and caspase 3) that were differentially expressed in PLum-AI cells compared to PLum-AD cells were subjected to biochemical validation by Western blotting. Biological process gene ontology (GO) analysis of the differentially expressed proteins demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to PI3 kinase and androgen receptor pathways. Besides, other relevant biological processes that are enriched in PLum-AI cells included cell adhesion and cell migration processes, cell and DNA damage, apoptosis, and cell cycle regulation. Conclusions: Our protein expression analysis of a murine in vitro model of PC progression identified differential protein spots that denote this progression and that comprise high-potential targets for early treatment of PC with a personalized patient-specific approach. Efforts are underway to functionally assess the potential roles of these proteins as therapeutic targets for PC progression.

Systematic biological and proteomics strategies to explore the regulation mechanism of Shoutai Wan on recurrent spontaneous Abortion's biological network

Ethnopharmacological relevanceShoutai Wan (STW) is a classic herbal formula for the treatment of recurrent spontaneous abortion (RSA), and clinical studies have shown the effectiveness of STW on RSA. However, the molecular mechanism of STW treatment of RSA is still unclear. Methods(1) Animal experiments: The normal pregnancy model was established with CBA/J*BALB/C, and the RSA model was established by CBA/J*DBA/2. The RSA model CBA/J*DBA/2 pregnant mice were randomly divided into four groups (RSA model group, STW low, medium and high dose groups) according to the order of pregnancy, respectively. The drug administration starts from the first day of pregnancy to the 14th day of pregnancy. The embryo loss rate (ELR) of each group was calculated. (2) Proteomic analysis of decidual tissue: The total protein of decidual tissue of each group was isolated by solid phase pH gradient 2-DE technique. The differentially expressed protein spots were analyzed and identified by PDQuest images; the peptide quality fingerprinting (PMF) was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Then, the proteins were identified by Mascot software searching, their functions were identified by bioinformatics strategy. (3) The expression of HSP27, α-enolase and Transferrin was detected by Western blotting and the expression of Annexin A2 and Transferrin was detected by immunohistochemistry. (4) The differential proteins and potential targets were analyzed by systematic biological strategy. Results(1) Compared with the normal group, the ELR in the RSA model group was significantly higher (P < 0.01). Compared with the model group, the ELR in the STW high, medium dose groups was lower (P < 0.01). (2) A 2-DE map of the decidual tissue of the RSA model group, normal pregnancy group, STW low, medium and high dose groups was established. Thirty proteins were identified. (3) The results of western blot showed that the expression of HSP27 and a-enolase in the RSA model group was higher than that in the normal group, and the expression of transferrin was lower (P < 0.01). Compared with the model group, the expression of HSP27 and a-enolase in STW high, medium dose groups was decreased (P < 0.01); Compared with the model group, the expression of Transferrin in the STW high dose group was increased (P < 0.01). (5) A lot of RSA treatment-related targets, biological processes and pathways were found after the systematic biological analysis. Conclusion(1) STW may reduce the ELR of the RSA mice. (2) The results of proteomics suggest that RSA is a complex process involving multiple proteins. STW can regulate the expression of various proteins in the decidual tissue of RSA mice, suggesting that it can act on multiple targets. (3) The results of western blotting of HSP27, a-enolase, transferrin were consistent with the results of proteomic analysis. (4) STW may achieve therapeutic effects by interfering with the targets, biological processes and signaling pathways discovered in this study.

Sarcomeric TPM3 expression in human heart and skeletal muscle.

In mammals, four tropomyosin genes TPM1, TPM2, TPM3, and TPM4 are known. One isoform of the TPM3 gene, encoding 285 amino acid residues designated as TPM3α, has been reported. TPM3α protein expression in human hearts is not definitively established. We have cloned from human heart and skeletal muscle transcripts of TPM3α and three novel TPM3 isoforms, TPM3ν, TPM3ξ, and TPM3ο. TPM3ν and TPM3ο are alternatively spliced RNAs with different 3'-UTRs encoding an identical novel protein with 285 amino acid differing from TPM3α and TPM3ξ in exon 6 only. TPM3α and TPM3ξ, which have different 3'UTRs, also encode an identical protein. qRT-PCR data show that the transcripts of TPM3α, TPM3ν, TPM3ξ, and TPM3ο are expressed in both heart and skeletal muscle. We have evaluated the expression of various TPM proteins in fetal and adult human hearts, and also in skeletal muscle samples. Western blots using CG3 antibody show a stronger signal of TPM3 protein in fetal heart and adult skeletal muscle compared to adult heart. LC-MS/MS studies with the protein spots separated and identified by CH1 antibody after 2D Western blot analyses, confirm the expression of TPM3α/TPM3ξ in heart, but some peptides detected could be either TPM3α or TPM3ν. In heart samples, TPM1 protein was the dominant with varying amount of TPM2 and TPM3, while TPM4 expression was not observed. In skeletal muscles, TPM2 was the majority TPM protein expressed. The biological consequences of these varying expression of individual tropomyosin proteins are yet to be established.

Impact of Sequential Passaging on Protein Expression of E. coli Using Proteomics Analysis.

Urinary tract infection (UTI) is one of the most prevalent bacterial infections in the world affecting the bladder and the kidney. Escherichia coli (E. coli) is the main causative agent of 80–90% of community-acquired UTIs, about 40% of nosocomial UTIs, and 25% of recurrent UTIs. The field of proteomics has emerged as a great tool to analyze expressed proteins to identify possible biomarkers associated with many pathological states and, to the same extent, those associated with bacterial pathogenesis and their ability to cause recurrent infections. Here, in a descriptive cross-sectional pilot study, we employed proteomic techniques to investigate the effects of environmental stress on protein profiles of E. coli simulated by sequential passaging of samples from patients with UTIs to screen for unique proteins that arise under stressful environment and could aid in the early detection of UTIs. Four urine samples were collected from individuals with recurrent UTI and sequentially subcultured; protein samples were extracted from bacterial pellets and analyzed using 2-dimensional gel electrophoresis (2DGE). Protein spots of interest arising from changes in the protein profile were analyzed using liquid chromatography-mass spectrometry (LC-MS/MS) and matched against known databases to identify related proteins. We identified ATPB_ECOBW, ASPA ECOLI, DPS ECOL6, and DCEB ECOLI as proteins associated with higher passaging. We concluded that passaging resulted in identifiable changes in the protein profile of E. coli, namely, proteins that are associated with survival and possible adaptation of bacteria, suggestive of factors contributing to antibiotic resistance and recurrent UTIs. Furthermore, our method could be further used to identify indicator-protein candidates that could be a part of a growing protein database to diagnose and identify causative agents in UTIs.

Effects of excess ammoniacal nitrogen (NH4+-N) on pigments, photosynthetic rates, chloroplast ultrastructure, proteomics, formation of reactive oxygen species and enzymatic activity in submerged plant Hydrilla verticillata (L.f.) Royle

Although excess ammoniacal-nitrogen (NH4+-N) results in the disturbance of various important biochemical and physiological processes, a detailed study on the effects of NH4+-N stress on the photosynthesis and global changes in protein levels in submerged macrophytes is still lacking. Here, the changes of excess NH4+-N on physiological parameters in Hydrilla verticillata (L.f.) Royle, a submerged macrophyte were investigated, including the contents of photosynthetic pigments, soluble sugars, net photosynthesis and respiration, glutamine synthetase (GS) and glutamate synthase (GOGAT) activities, chloroplast ultrastructure, chloroplast reactive oxygen species (ROS) accumulation and protein levels. Our results showed that the net photosynthetic rate and pigment content reached maximum values when the plants were treated with 1 and 2 mg L-1 NH4+-N, respectively, and decreased at NH4+-N concentrations at 5, 10, 15 and 20 mg L-1. This decrease might be caused by ROS accumulation. Compared that in 0.02 mg L-1 NH4+-N as a control, ROS generation in chloroplasts significantly increased in the presence of more than 2 mg L-1 NH4+-N. Consistently, the damages caused by over-accumulated ROS were observed in chloroplast ultrastructure, showing a loose thylakoid membranes and swollen grana/stroma lamellae. Furthermore, through proteomic analysis, we identified 91 differentially expressed protein spots. Among them, six proteins involved in photosynthesis decreased in abundance in response to excess NH4+-N. Surprisingly, the abundance of all the identified proteins that were involved in nitrogen assimilation and amino acid metabolism tended to increase under excess NH4+-N compared with the control, suggestive of the imbalanced carbon and nitrogen (C-N) metabolisms. In support, activated GS and GOGAT cycle was observed, evidenced by higher activities of GS and GOGAT enzymes. To our knowledge, this work is the first description that excess NH4+-N results in chloroplast ultrastructural damages and the first proteomic evidence to support that excess NH4+-N can lead to a decline in photosynthesis and imbalance of C-N metabolism in submerged macrophytes.

Proteomic comparison of non-sexed and sexed (X-bearing) cryopreserved bull semen

It is widely recognized that quality of spermatozoa in sexed semen (SS) samples is not as great as for conventional, non-sexed semen (NS). There are differences in qualitative and biochemical variables between spermatozoa in NS and SS. Information, however, is lacking on molecular differences, especially concerning spermatozoa proteomic differences is SS and NS. The objective of this study was to compare commercially available NS and SS bull semen by evaluating sperm quality variables in conjunction with a proteomics approach. Results from flow cytometry and computer-assisted sperm analyses indicated there was less sperm motility, viability, mitochondrial potential and acrosome integrity in sperm from SS. Results from proteomic analysis indicated sperm from NS and SS samples were characterized by different protein profiles. There was identification of 70 sperm proteins that differed in abundance and six protein spots that were different in extent of carbonylation. Sperm from SS had altered structures of enzymes involved in glycolysis, oxidative phosphorylation and maintenance of a constant adenylate energy charge. Furthermore, sperm from SS had alterations of several flagella substructures, especially outer dense fiber proteins, which were less abundant and more carbonylated than in sperm from NS. In sperm of SS, compared with NS, there were differences in abundance of proteins involved in capacitation, acrosome reaction and sperm-egg fusion as well as lesser abundances of sperm surface proteins. Results enable a greater understanding of differences between sperm from NS and SS samples, thereby contributing to development of improved protocols for more effective protection of sexed spermatozoa during processing.

Differences in proteome response to cold acclimation in Zoysia japonica cultivars with different levels of freeze tolerance

AbstractZoysiagrasses (Zoysia spp.) are warm‐season turfgrasses primarily grown in the southern and transition zones of the United States. An understanding of the physiological and proteomic changes that zoysiagrasses undergo during cold acclimation may shed light on phenotypic traits and proteins useful in selection of freeze‐tolerant genotypes. We investigated the relationship between cold acclimation, protein expression, and freeze tolerance in cold acclimated (CA) and nonacclimated (NA) plants of Zoysia japonica Steud. cultivars Meyer (freeze‐tolerant) and Victoria (freeze‐susceptible). Meristematic tissues from the grass crowns were harvested for proteomic analysis. Freeze testing indicated that cold acclimation accounted for a 1.9‐fold increase in plant survival than nonacclimation treatment. Overall, proteomic analysis identified 62 protein spots differentially accumulated in abundance under cold acclimation. Nine and 22 unique protein spots were identified for Meyer and Victoria, respectively, with increased abundance or decreased abundance. In addition, 23 shared protein spots were found among the two cultivars in response to cold acclimation. Function classification revealed that these proteins were involved primarily in transcription, signal transduction and stress defense, carbohydrate and energy metabolism, and protein and amino acid metabolism. Several proteins of interest for their association with cold acclimation were identified. Further investigation of these proteins and their functional categories may contribute to increase our understanding of the differences in freezing tolerance among zoysiagrass germplasm.

The Identification of Native Epitopes Eliciting a Protective High-Affinity Immunoglobulin Subclass Response to Blood Stages of Plasmodium falciparum: Protocol for Observational Studies.

BackgroundAntibodies to blood stages protective against complications of Plasmodium falciparum infection were found to be of immunoglobulin G 1 (IgG1) and IgG3 subclasses and of high affinity to the target epitopes. These target epitopes cannot be characterized using recombinant antigens because of a lack of appropriate glycosylation, phosphorylation, methylation, and bisulfide bond formation, which determine the structure of conformational and nonlinear epitopes within the tertiary and quaternary structures of native P. falciparum antigens.ObjectiveThis study aims to develop a method for the comprehensive detection of all P. falciparum schizont antigens, eliciting a protective immune response.MethodsPurified parasitophorous vacuole membrane–enclosed merozoite structures (PEMSs) containing native schizont antigens are initially generated, separated by two-dimensional (2D) gel electrophoresis and blotted onto nitrocellulose. Antigens eliciting a protective antibody response are visualized by incubation with sera from patients with clinical immunity. This is followed by the elution of low-affinity antibodies with urea and detection of protective antibody responses by incubation with anti-IgG1 and anti-IgG3 antibodies, which were conjugated to horseradish peroxidase. This is followed by visualization with a color reaction. Blot signals are normalized by relating to the intensity of blot staining with a reference antibody and housekeeping antigens. Results are corrected for intensity of exposure by the relation of antibody responses to global P. falciparum antibody titers. Antigens eliciting the protective responses are identified as immunorelevant from the comparison of spot positions, indicating high-affinity IgG1 or IgG3 responses on the western blot, which is unique to or consistently more intensive in clinically immune individuals compared with nonimmune individuals. The results obtained are validated by using affinity chromatography.ResultsAnother group previously applied 2D western blotting to analyze antibody responses to P. falciparum. The sera of patients allowed the detection of 42 antigenic spots on the 2D immunoblot. The spots detected were excised and subjected to mass spectrometry for identification. A total of 19 protein spots were successfully identified and corresponded to 13 distinct proteins. Another group used immunoaffinity chromatography to identify antigens bound by IgGs produced by mice with enhanced immunity to Plasmodium yoelii. Immunorelevant antigens were isolated and identified by immobilizing immunoglobulin from immune mice to a Sephadex column and then passing a blood-stage antigen mixture through the column followed by the elution of specific bound antigens with sodium deoxycholate and the identification of those antigens by western blotting with specific antibodies.Conclusions2D western blotting using native antigens has the potential to identify antibody responses selective for specific defined isomeric forms of the same protein, including isoforms (protein species) generated by posttranscriptional modifications such as phosphorylation, glycosylation, and methylation. The process involved in 2D western blotting enables highly sensitive detection, high resolution, and preservation of antibody responses during blotting. Validation by immunoaffinity chromatography can compensate for the antigen loss associated with the blotting process. It has the potential for indirect quantification of protective antibody responses by enabling quantification of the amount of eluted antibody bound antigens through mass spectrometry.International Registered Report Identifier (IRRID)PRR1-10.2196/15690

Study on Effect of Extraction Techniques and Seed Coat on Proteomic Distribution and Cheese Production from Soybean Milk.

Soybean-based food products are a major source of protein. In the present study, proteins in soybean milk from seeds of the cultivar Bunya (Glycine max) were extracted using the cheesecloth and the centrifuge methods. The milk was produced through mechanical crushing of both whole and split seeds in water. Following separation by either the cheesecloth or centrifuge, proteins were isolated from the soybean milk by using thiourea/urea solubilisation and then separated them using two-dimensional polyacrylamide gel electrophoresis. The isolated proteins were identified by mass spectrometry. A total of 97 spots were identified including 49 that displayed different abundances. Of the two separation techniques, centrifuge separation gave higher protein extraction and more intense protein spots than cheesecloth separation. Eleven of the β-subunits of β-conglycinin, three of the α-subunits of β-conglycinin, and four of the mutant glycinin showed different levels of abundances between separation techniques, which might be related to subsequent cheese quality. Notably, split-seed soybean milk has less allergenic proteins with four α-subunits of β-conglycinin compared to whole-seed milk with eight of those proteins. The sensory evaluation showed that the cheese produced from split-soybean milk received higher consumer preferences compared to that of whole seed, which could be explained by their proteomic differences. The demonstrated reference map for whole and split-seed soybean milk could be further utilized in the research related to soybean cheesemaking.

Two Dimensional Gel Electrophoresis Image Inpainting for Improving Protein Spot Detection

Two Dimensional Gel Electrophoresis Image Inpainting for Improving Protein Spot Detection

Comparative Proteomics and Secretomics Revealed Virulence and Antibiotic Resistance-Associated Factors in Vibrio parahaemolyticus Recovered From Commonly Consumed Aquatic Products.

Vibrio parahaemolyticus is a seafoodborne pathogen that can cause severe gastroenteritis and septicemia diseases in humans and even death. The emergence of multidrug-resistant V. parahaemolyticus leads to difficulties and rising costs of medical treatment. The bacterium of environmental origins containing no major virulence genes (tdh and trh) has been reported to be associated with infectious diarrhea disease as well. Identification of risk factors in V. parahaemolyticus is imperative for assuming food safety. In this study, we obtained secretomic and proteomic profiles of V. parahaemolyticus isolated from 12 species of commonly consumed aquatic products and identified candidate protein spots by using two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry techniques. A total of 11 common and 28 differential extracellular proteins were found from distinct secretomic profiles, including eight virulence-associated proteins: outer membrane channel TolC, maltoporin, elongation factor Tu, enolase, transaldolase, flagellin C, polar flagellin B/D, and superoxide dismutase, as well as five antimicrobial and/or heavy metal resistance-associated ABC transporter proteins. Comparison of proteomic profiles derived from the 12 V. parahaemolyticus isolates also revealed five intracellular virulence-related proteins, including aldehyde-alcohol dehydrogenase, outer membrane protein A, alkyl hydroperoxide reductase C, phosphoenolpyruvate-protein phosphotransferase, and phosphoglycerate kinase. Additionally, our data indicated that aquatic product matrices significantly altered proteomic profiles of the V. parahaemolyticus isolates with a number of differentially expressed proteins identified. The results in this study meet the increasing need for novel diagnosis candidates of the leading seafoodborne pathogen worldwide.