Changes in protein expression in the sheep abomasum following trickle infection with Teladorsagia circumcincta

Parasitology ◽  
2011 ◽  
Vol 139 (3) ◽  
pp. 375-385 ◽  
Author(s):  
ALAN D. PEMBERTON ◽  
JEREMY K. BROWN ◽  
NICKY M. CRAIG ◽  
JUDITH PATE ◽  
KEVIN McLEAN ◽  
...  
Keyword(s):  
Western Blotting ◽  
Peptide Mass Fingerprinting ◽  
Tissue Samples ◽  
Host Protection ◽  
Teladorsagia Circumcincta ◽  
Peptide Mass ◽  
Proteomic Approach ◽  
2D Gel ◽  
Albumin Mrna ◽  
Parasite Challenge

SUMMARYContinual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to naïve sheep, as potential key mediators of immunity. Six naïve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the naïve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.

scholarly journals Hyperoxia-Induced Protein Alterations in Renal Rat Tissue: A Quantitative Proteomic Approach to Identify Hyperoxia-Induced Effects in Cellular Signaling Pathways

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Jochen Hinkelbein ◽  
Lennert Böhm ◽  
Oliver Spelten ◽  
David Sander ◽  
Stefan Soltész ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rat Kidney ◽  
Peptide Mass Fingerprinting ◽  
Renal Tissue ◽  
Short Term ◽  
Two Dimensional Gel Electrophoresis ◽  
Normobaric Hyperoxia ◽  
Peptide Mass ◽  
Proteomic Approach ◽  
Hyperoxia Exposure

Introduction. In renal tissue as well as in other organs, supranormal oxygen pressure may lead to deleterious consequences on a cellular level. Additionally, hyperoxia-induced effect in cells and related free radicals may potentially contribute to renal failure. The aim of this study was to analyze time-dependent alterations of rat kidney protein expression after short-term normobaric hyperoxia using proteomics and bioinformatic approaches.Material and Methods.N=36Wistar rats were randomized into six different groups: three groups with normobaric hyperoxia (exposure to 100% oxygen for 3 h) and three groups with normobaric normoxia (NN; room air). After hyperoxia exposure, kidneys were removed immediately, after 3 days and after 7 days. Kidney lysates were analyzed by two-dimensional gel electrophoresis followed by peptide mass fingerprinting using tandem mass spectrometry. Statistical analysis was performed with DeCyder 2D software (p<0.01). Biological functions of differential regulated proteins were studied using functional network analysis (Ingenuity Pathways Analysis and PathwayStudio).Results. Expression of 14 proteins was significantly altered(p<0.01): eight proteins (MEP1A_RAT, RSSA_RAT, F16P1_RAT, STML2_RAT, BPNT1_RAT, LGMN_RAT, ATPA_RAT, and VDAC1_RAT) were downregulated and six proteins (MTUS1_RAT, F16P1_RAT, ACTG_RAT, ACTB_RAT, 2ABA_RAT, and RAB1A_RAT) were upregulated. Bioinformatic analyses revealed an association of regulated proteins with inflammation.Conclusions. Significant alterations in renal protein expression could be demonstrated for up to 7 days even after short-term hyperoxia. The identified proteins indicate an association with inflammation signaling cascades. MEP1A and VDAC1 could be promising candidates to identify hyperoxic injury in kidney cells.

scholarly journals A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins

2003 ◽  
Vol 371 (2) ◽  
pp. 331-340 ◽  
Author(s):  
Jean-Philippe GAGNÉ ◽  
Joanna M. HUNTER ◽  
Benoît LABRECQUE ◽  
Benoît CHABOT ◽  
Guy G. POIRIER
Keyword(s):  
Protein Identification ◽  
Binding Proteins ◽  
Peptide Mass Fingerprinting ◽  
Sequence Motif ◽  
Dot Blot ◽  
New Family ◽  
Peptide Mass ◽  
Proteomic Approach ◽  
Heterogeneous Nuclear Ribonucleoproteins ◽  
Binding Sequence

A new class of poly(ADP-ribose) (pADPr)-binding proteins, heterogeneous nuclear ribonucleoproteins (hnRNPs), has been identified by a proteomic approach using matrix-assisted laser-desorption–ionization time-of-flight ('MALDI-TOF') MS. Liquid-phase isoelectric focusing with a Rotofor® cell (Bio-Rad) allowed pre-fractionation of proteins extracted from HeLa cells. Rotofor® protein fractions were further separated by SDS/PAGE and then transferred to a PVDF membrane. pADPr-binding proteins were analysed by autoradiography of the protein blot after incubation with 32P-labelled automodified pADPr polymerase-1 (PARP-1). Peptide mass fingerprinting of selected bands identified the most abundant pADPr-binding proteins as hnRNPs, a family of proteins that bind pre-mRNA into functional complexes involved in mRNA maturation and transport to the cytoplasm. Sequence homology database searching against a previously reported pADPr-binding sequence motif revealed that the hnRNPs contain a putative pADPr-binding sequence pattern [Pleschke, Kleczkowska, Strohm and Althaus (2000) J. Biol. Chem. 275, 40974–40980]. pADPr-binding assays performed with synthetic peptides by the dot-blot technique and with nitrocellulose-transferred recombinant hnRNPs confirmed the pADPr-binding protein identification and the specificity of the interaction. These results could establish a link between increased levels of pADPr in DNA damaged cells and the modified protein expression pattern resulting from altered mRNA trafficking.

Abstract 89: Mitochondrial Protein Acetylation in Aged Hearts in the Context of Ischemia-Reperfusion Injury

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Sergiy M Nadtochiy ◽  
William R Urciuoli ◽  
Paul S Brookes ◽  
George A Porter
Keyword(s):  
Western Blot ◽  
Complex I ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Mitochondrial Protein ◽  
Peptide Mass Fingerprinting ◽  
Protein Acetylation ◽  
Post Translational Modification ◽  
Peptide Mass ◽  
2D Gel

Introduction: SIRT3 deficiency leads to increased mitochondrial acetylation, and enhances sensitivity of the heart to stress. Recently we demonstrated that a worse outcome of ischemia-reperfusion (IR) injury in aged hearts was associated with increased protein acetylation. The aim of this study was to investigate the mechanism of acetylation and the identity of acetylated targets in aged hearts in the context of IR injury. Methods: Mitochondria were isolated from WT adult (7 mo.), SIRT3 +/- adult (7 mo.) and WT aged (18 mo.) hearts. SIRT3 protein expression and identification of acetylated proteins from SIRT3 +/- adult and WT aged samples were investigated using proteomics (2D gel, western blot, peptide mass fingerprinting). Results: Mitochondrial acetylome analysis revealed that many hyperacetylated patterns were identical between adult SIRT3 +/- and aged WT hearts, suggesting that increased protein acetylation in the aged WT heart might be due to SIRT3 inhibition. Several targets were indentified, including complex I NADH dehydrogenase flavoprotein 1, which is known to interact with SIRT3. In this regard, we demonstrated that respiratory complex I (CxI) activity was significantly inhibited in both SIRT3 +/- adult and WT aged hearts. Analysis of SIRT3 protein revealed that negatively charged species of SIRT3 (relative to the bulk SIRT3 population), were lost in aged hearts. These alterations suggest post-translational modification of SIRT3 which may lead to loss of its deacetylase activity. Alternatively, we found that upregulation of the mitochondrial acetyltransferase GCN5L1 in aged hearts (examined by western blot) may also contribute to enhanced protein acetylation. Conclusions: These data support a connection between SIRT3 downregulation, mitochondrial protein acetylation, and exacerbation of IR injury in aging hearts. Furthermore, it is known that functional CxI is required for several modes of cardioprotection suggesting that downregulation of CxI in SIRT3 +/- or WT aged hearts may underlie their poor recovery from IR injury.

Proteomic Analysis of Medicinal Plant Calotropis Gigantea by insilico Peptide Mass Fingerprinting

2020 ◽  
Vol 16 ◽  
Author(s):  
Saad Ur Rehman ◽  
Muhammad Rizwan ◽  
Sajid Khan ◽  
Azhar Mehmood ◽  
Anum Munir
Keyword(s):  
Medicinal Plant ◽  
Structure Prediction ◽  
3D Structure ◽  
Peptide Mass Fingerprinting ◽  
Protein Docking ◽  
Receptor Protein ◽  
Human Leukemia ◽  
Protein Database ◽  
Calotropis Gigantea ◽  
Peptide Mass

: Medicinal plants are the basic source of medicinal compounds traditionally used for the treatment of human diseases. Calotropis gigantea a medicinal plant belonging to the family of Apocynaceae in the plant kingdom and subfamily Asclepiadaceae usually bearing multiple medicinal properties to cure a variety of diseases. Background: The Peptide Mass Fingerprinting (PMF) identifies the proteins from a reference protein database by comparing the amino acid sequence that is previously stored in a database and identified. Method: The calculation of insilico peptide masses is done through the ExPASy PeptideMass and these masses are used to identify the peptides from MASCOT online server. Anticancer probability is calculated from the iACP server, docking of active peptides is done by CABS-dock the server. Objective: The purpose of the study is to identify the peptides having anti-cancerous properties by in-silico peptide mass fingerprinting. Results : The anti-cancerous peptides are identified with the MASCOT peptide mass fingerprinting server, the identified peptides are screened and only the anti-cancer are selected. De novo peptide structure prediction is used for 3D structure prediction by PEP-FOLD 3 server. The docking results confirm strong bonding with the interacting amino acids of the receptor protein of breast cancer BRCA1 which shows the best peptide binding to the Active chain, the human leukemia protein docking with peptides shows the accurate binding. Conclusion : These peptides are stable and functional and are the best way for the treatment of cancer and many other deadly diseases.

scholarly journals The Fish Pathogen Vibrio ordalii Under Iron Deprivation Produces the Siderophore Piscibactin

2019 ◽  
Vol 7 (9) ◽  
pp. 313 ◽  
Author(s):  
Pamela Ruiz ◽  
Miguel Balado ◽  
Juan Carlos Fuentes-Monteverde ◽  
Alicia E. Toranzo ◽  
Jaime Rodríguez ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Iron Uptake ◽  
Peptide Mass Fingerprinting ◽  
Iron Limitation ◽  
Fish Pathogen ◽  
Fish Pathogens ◽  
Iron Deprivation ◽  
Peptide Mass ◽  
Photobacterium Damselae ◽  
Vibrio Ordalii

Vibrio ordalii is the causative agent of vibriosis, mainly in salmonid fishes, and its virulence mechanisms are still not completely understood. In previous works we demonstrated that V. ordalii possess several iron uptake mechanisms based on heme utilization and siderophore production. The aim of the present work was to confirm the production and utilization of piscibactin as a siderophore by V. ordalii. Using genetic analysis, identification by peptide mass fingerprinting (PMF) of iron-regulated membrane proteins and chemical identification by LC-HRMS, we were able to clearly demonstrate that V. ordalii produces piscibactin under iron limitation. The synthesis and transport of this siderophore is encoded by a chromosomal gene cluster homologous to another one described in V. anguillarum, which also encodes the synthesis of piscibactin. Using β-galactosidase assays we were able to show that two potential promoters regulated by iron control the transcription of this gene cluster in V. ordalii. Moreover, biosynthetic and transport proteins corresponding to piscibactin synthesis and uptake could be identified in membrane fractions of V. ordalii cells grown under iron limitation. The synthesis of piscibactin was previously reported in other fish pathogens like Photobacterium damselae subsp. piscicida and V. anguillarum, which highlights the importance of this siderophore as a key virulence factor in Vibrionaceae bacteria infecting poikilothermic animals.

scholarly journals Optimization of Protein Extraction Method for 2DE Proteomics of Goat’s Milk

Molecules ◽  
2020 ◽  
Vol 25 (11) ◽  
pp. 2625
Author(s):  
Muzammeer Mansor ◽  
Jameel R. Al-Obaidi ◽  
Nurain Nadiah Jaafar ◽  
Intan Hakimah Ismail ◽  
Atiqah Farah Zakaria ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Extraction ◽  
Chloroform Solution ◽  
Milk Proteins ◽  
Peptide Mass Fingerprinting ◽  
Extraction Methods ◽  
Dairy Goats ◽  
Goat’S Milk ◽  
Peptide Mass ◽  
Goat's Milk

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat’s milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat’s milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat’s milk proteomes and the complexity of goat’s milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat’s milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat’s milk.

scholarly journals Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears

2002 ◽  
Vol 366 (2) ◽  
pp. 511-520 ◽  
Author(s):  
Benjamin L. SCHULZ ◽  
David OXLEY ◽  
Nicolle H. PACKER ◽  
Niclas G. KARLSSON
Keyword(s):  
Molecular Mass ◽  
Peptide Mass Fingerprinting ◽  
High Molecular Mass ◽  
Tear Fluid ◽  
Protein Variant ◽  
Peptide Mass ◽  
Composite Gel ◽  
Molecular Masses ◽  
Protein Components ◽  
Sialyl Lea

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.

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