Proteomic Changes of Porcine Oocytes After Vitrification and Subsequent in vitro Maturation: A Tandem Mass Tag-Based Quantitative Analysis
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ABSTRACT:
SUBMITTER: Jia B
PROVIDER: S-EPMC7785821 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:Porcine deltacoronavirus (PDCoV) is a newly emerging porcine pathogenic enteric coronavirus that can cause diarrhea, vomiting, dehydration, and a high mortality rate in piglets. At present, the understanding of PDCoV pathogenesis is very limited, which seriously hinders effective prevention and control. In this study, liquid chromatography tandem-mass spectrometry (LC-MS/MS) combined with tandem mass tag (TMT) labeling was performed to compare the differential expression of proteins in PDCoV-infected and mock-infected LLC-PK cells at 18 h post-infection (hpi). In addition, the parallel reaction monitoring (PRM) technique was used to verify the quantitative proteome data. A total of 4624 differentially expressed proteins (DEPs) were quantitated, of which 128 were significantly upregulated, and 147 were significantly downregulated. Bioinformatics analysis revealed that these DEPs were involved mainly in the defense response, apoptosis, and the immune system, and several DEPs may be related to interferon-stimulated genes and the immune system. Based on DEP bioinformatics analysis, we propose that PDCoV infection may utilize the apoptosis pathway of host cells to achieve maximum viral replication. Meanwhile, the host may be able to stimulate the transcription of interferon-stimulated genes (ISGs) through the JAK/STAT signaling pathway to resist the virus. Overall, in this study, we presented the first application of proteomics analysis to determine the protein profile of PDCoV-infected cells, which provides valuable information with respect to better understanding the host response to PDCoV infection and the specific pathogenesis of PDCoV infection.
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Project description:Parasitic isopods perforate and attach to the host integument via the mandibles and then feed on hemolymph and exudate from the wounds. Such isopods attack a variety of commercially important fish and crustacean hosts. Similar to other hematophagous parasites, isopods may also employ biomolecules that affect host blood conglutination and defense systems. In the present study, a tandem mass tag-based quantitative proteomic approach was used to identify differentially expressed proteins in Tachaea chinensis parasites of shrimp, by comparing parasitic (fed) and pre-parasitic (unfed) individuals. We identified 888 proteins from a total of 1,510 peptides, with a significant difference in 129 between the fed and unfed groups. Among these, 37 were upregulated and 92 were downregulated in unfed T. chinensis. This indicates that T. chinensis may require more energy before parasitism during its search for a host. In addition, as is the case for other blood-sucking parasites, it might secrete antihemostatic, anti-inflammatory, and immunomodulatory molecules to facilitate blood meal acquisition. To our knowledge, this study is the first to use a TMT-based proteomic approach to analyze the proteome of isopod parasites, and the results will facilitate our understanding of the molecular mechanisms of isopod parasitism on crustaceans.
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Project description:Background and aimSpecific tumor biomarkers are useful for the early diagnosis of cancer or can predict the recurrence of neoplastic disease in humans and animals. Lymphoma in dogs could be classified into B-, T-, and NK-cell origins. T-cell lymphoma has the worst prognosis with a shorter survival time and disease-free interval. This study aimed to identify the differential serum protein expressions of canine B- and T-cell lymphomas compared with healthy dogs using a tandem mass tag (TMT)-based quantitative proteomics.Materials and methodsSerum samples were collected from 20 untreated canine lymphomas (14 B-cells and 6 T-cells) and four healthy control dogs. Sera peptides from each sample were processed for TMT 10-plex tagging and analyzed using liquid chromatography-mass spectrometry (MS). Differential proteome profiling was then compared between lymphoma and control.ResultsWe discovered 20 elevated and 14 decreased serum proteins in the lymphoma group relative to the healthy group. Six candidate increased proteins in canine lymphomas were beta-actin cytoplasmic 1 (ACTB, p=0.04), haptoglobin (p=0.002), beta-2 microglobulin (aaaaaaaa2M, p=0.007), beta-2 glycoprotein 1 (APOH, p=0.03), metalloproteinase inhibitor 1 (TIMP-1, p=0.03), and CD44 antigen (p=0.02). When compared between B- and T-cell lymphomas, B-cell phenotypes had upregulated immunoglobulin (Ig) heavy chain V region GOM (p=0.02), clusterin (p=0.01), apolipoprotein C1 (APOC1, p=0.05), and plasminogen (p=0.02).ConclusionThese findings were investigated quantitative serum proteomes between B- and T-cell lymphomas using TMT-based MS. ACTB, aaaaaaaa2M, APOH, TIMP-1, CD44 antigen, Ig heavy chain V region GOM, and APOC1 are novel candidate proteins and might serve as a lymphoma biomarker in dogs. However, evaluation with an increased sample size is needed to confirm their diagnostic and prognostic ability.
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Project description:Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies.
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Project description:Reticuloendotheliosis virus (REV) is a type C avian retrovirus that causes immunosuppression, dwarf syndrome, and lymphoma in infected hosts. In this study, we used tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to characterize protein alterations in chicken bursa of Fabricius, before and after REV infection at 7, 14, 21, and 28 days. Our data showed that 1,127, 999, 910, and 1,138 differentially expressed proteins were significantly altered at 7, 14, 21, and 28 days after REV infection, respectively. Morphological analysis showed that REV infection reduced in cortical lymphocytes, bursal follicle atrophy, and nuclear damage. Bioinformatics analysis indicated these proteins were mainly involved with immune responses, energy metabolism, cellular processes, biological regulation, metabolic processes, response to stimuli, and multicellular organismal process. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway cluster analysis showed that post-infection, proteins were enriched in the cell cycle, Wnt signaling, antigen processing and presentation, cytokine receptor interaction, adenosine 3',5'-cyclic monophosphate signaling pathway, and NF-κB signaling. In addition, we observed that peroxiredoxin 4 (PRDX4), peroxiredoxin 6 (PRDX6), glutathione peroxidase 3 (GPX3), catalase (CAT), and peroxidasin (PXDN) were involved in oxidative stress. Some heat shock protein (HSP) family members such as HSPH1, DNAJA4, HSPA8, and HSPA4L also changed significantly after REV infection. These findings help clarify interactions between REV and the host and provides mechanistic insights on REV-induced host immunosuppression.
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Project description:Esophageal cancer (EC) is a type of extremely aggressive gastrointestinal cancer with high incidences in China and other Asian countries. EC does not have specific symptoms and is relatively easy to metastasize, which makes it difficult in early diagnosis. Thus, novel noninvasive diagnostic method is urgently needed in clinical practice. In this study, mass spectrometry with tandem mass tags and differential protein analysis were applied for identifying esophageal cancer-related proteins. The identified proteins were annotated based on their enrichment in Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. In addition, hierarchical clustering was applied based on differentially expressed proteins. As a result, a total of 5131 quantifiable proteins were identified from our liquid chromatography-tandem mass spectrometry with tandem mass tags (LC-MS/MS-TMT) method with 63 upregulated and 97 downregulated differential proteins between esophageal cancer and controlled normal samples. The differentially expressed proteins were highly enriched in GO terms associated with mitochondrial dissemble and apoptosis, and blood vessel regulation, and the upregulated differentially expressed proteins in EC samples were significantly enriched in major histocompatibility complex MHC-class I/II pathway of immune system. The functional clustering analysis revealed potential protein-protein interactions among tetraspanin, myosin, and S-100. In summary, our study provided a practical technological procedure of proteomic analysis for discovering novel biomarkers of a specific cancer type.
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Project description:Highly pathogenic H5N1 influenza viruses (HPAIV) cause rapid systemic illness and death in susceptible animals, leading to a disease with high morbidity and mortality rates. Although vaccines and drugs are the best solution to prevent this threat, a more effective treatment for H5 strains of influenza has yet to be developed. Therefore, the development of therapeutics/drugs that combat H5N1 influenza virus infection is becoming increasingly important. Lycorine, the major component of Amaryllidaceae alkaloids, exhibits better protective effects against A/CK/GD/178/04 (H5N1) (GD178) viruses than the commercial neuraminidase (NA) inhibitor oseltamivir in our prior study. Lycorine demonstrates outstanding antiviral activity because of its inhibitory activity against the export of viral ribonucleoprotein complexes (vRNPs) from the nucleus. However, how lycorine affects the proteome of AIV infected cells is unknown. Therefore, we performed a comparative proteomic analysis to identify changes in protein expression in AIV-infected Madin-Darby Canine Kidney cells treated with lycorine. Three groups were designed: mock infection group (M), virus infection group (V), and virus infection and lycorine-treated after virus infection group (L). The multiplexed tandem mass tag (TMT) approach was employed to analyze protein level in this study. In total, 5,786 proteins were identified from the three groups of cells by using TMT proteomic analysis. In the V/M group, 1,101 proteins were identified, of which 340 differentially expressed proteins (DEPs) were determined during HPAIV infection; among the 1,059 proteins identified from the lycorine-treated group, 258 proteins presented significant change. Here, 71 proteins showed significant upregulation or downregulation of expression in the virus-infected/mock and virus-infected/lycorine-treated comparisons, and the proteins in each fraction were functionally classified further. Interestingly, lycorine treatment decreased the levels of the nuclear pore complex protein 93 (Nup93, E2RSV7), which is associated with nuclear-cytoplasmic transport. In addition, Western blot experiments confirmed that the expression of Nup93 was significantly downregulated in lycorine treatment but induced after viral infection. Our results may provide new insights into how lycorine may trap vRNPs in the nucleus and suggest new potential therapeutic targets for influenza virus.
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Project description:Wheat (Triticum aestivum L.) is an important crop worldwide. The physiological deterioration of seeds during storage and seed priming is closely associated with germination, and thus contributes to plant growth and subsequent grain yields. In this study, wheat seeds during different stages of artificial ageing (45°C; 50% relative humidity; 98%, 50%, 20%, and 1% Germination rates) and priming (hydro-priming treatment) were subjected to proteomics analysis through a proteomic approach based on the isobaric tandem mass tag labeling. A total of 162 differentially expressed proteins (DEPs) mainly involved in metabolism, energy supply, and defense/stress responses, were identified during artificial ageing and thus validated previous physiological and biochemical studies. These DEPs indicated that the inability to protect against ageing leads to the incremental decomposition of the stored substance, impairment of metabolism and energy supply, and ultimately resulted in seed deterioration. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the up-regulated proteins involved in seed ageing were mainly enriched in ribosome, whereas the down-regulated proteins were mainly accumulated in energy supply (starch and sucrose metabolism) and stress defense (ascorbate and aldarate metabolism). Proteins, including hemoglobin 1, oleosin, agglutinin, and non-specific lipid-transfer proteins, were first identified in aged seeds and might be regarded as new markers of seed deterioration. Of the identified proteins, 531 DEPs were recognized during seed priming compared with unprimed seeds. In contrast to the up-regulated DEPs in seed ageing, several up-regulated DEPs in priming were involved in energy supply (tricarboxylic acid cycle, glycolysis, and fatty acid oxidation), anabolism (amino acids, and fatty acid synthesis), and cell growth/division. KEGG and protein-protein interaction analysis indicated that the up-regulated proteins in seed priming were mainly enriched in amino acid synthesis, stress defense (plant-pathogen interactions, and ascorbate and aldarate metabolism), and energy supply (oxidative phosphorylation and carbon metabolism). Therefore, DEPs associated with seed ageing and priming can be used to characterize seed vigor and optimize germination enhancement treatments. This work reveals new proteomic insights into protein changes that occur during seed deterioration and priming.
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Project description:Mass spectrometry (MS) coupled toisobaric labeling has developed rapidly into a powerful strategy for high-throughput protein quantification. Sample multiplexing and exceptional sensitivity allow for the quantification of tens of thousands of peptides and, by inference, thousands of proteins from multiple samples in a single MS experiment. Accurate quantification demands a consistent and robust sample-preparation strategy. Here, we present a detailed workflow for SPS-MS3-based quantitative abundance profiling of tandem mass tag (TMT)-labeled proteins and phosphopeptides that we have named the streamlined (SL)-TMT protocol. We describe a universally applicable strategy that requires minimal individual sample processing and permits the seamless addition of a phosphopeptide enrichment step ("mini-phos") with little deviation from the deep proteome analysis. To showcase our workflow, we profile the proteome of wild-type Saccharomyces cerevisiae yeast grown with either glucose or pyruvate as the carbon source. Here, we have established a streamlined TMT protocol that enables deep proteome and medium-scale phosphoproteome analysis.
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Project description:Currently, determining when to start antiviral therapy in patients with chronic HBV infection is a controversial issue. One crucial reason is that biomarkers for distinguishing the natural history of chronic HBV infection are unmet needs. In this study, we aimed to explore novel biomarkers and therapeutic targets for the diagnosis and treatment of chronic HBV infection by using tandem mass tag (TMT)-based quantitative proteomics approach. Here, we firstly revealed the serum proteomic characterization of the natural history of chronic HBV infection using multiplex TMT labeling coupled with liquid chromatography-mass spectrometry. Then, we verified the levels of differentially expressed proteins (DEPs) across a large number of clinical samples by enzyme-linked immunosorbent assay (ELISA). We found that DEPs over the different phases of chronic HBV infection were primarily involved in the biological process of leukocyte-mediated immunity. Patients with chronic hepatitis were characterized as having an up-regulated proteasome pathway, including upregulation of proteasome activator subunit 1 (PSME1) and proteasome subunit alpha type 7 (PSMA7) levels. In addition, immune tolerant phase patients were characterized by having the lowest ephrin-B2 (EFNB2) levels and highest heat responsive protein 12 (HRSP12) levels. Moreover, inactive HBV carrier state patients were characterized by having a down-regulated glycolysis/gluconeogenesis pathway, with especially low expression of related enzymes alpha-enolase (ENO1) and fructose-1,6-bisphosphatase 1 (FBP1). What's more, HBeAg-negative chronic hepatitis patients were characterized as having the highest interleukin 18 binding protein (IL-18BP) levels. Thus, our results provide several potential diagnostic biomarkers for distinguishing the natural history of chronic HBV infection, such as PSME1, PSMA7, EFNB2, ENO1, and IL-18BP, and also present potential therapeutic interventions for chronic hepatitis B patients, such as targeting the proteasome or glycolysis/gluconeogenesis pathways. Our findings shed new light on the development of novel diagnostic biomarkers and therapeutic targets for the diagnosis and treatment of chronic HBV infection.
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