Nogo-B co-IP-MS experiment in mouse liver and pancreas
Ontology highlight
ABSTRACT: Co-immunoprecipitation with either rabbit normal IgG or anti-Nogo-B antibody followed by mass spectrometry (co-IP-MS) in the liver and pancreas of wild-type C57BL/6J mice.
Project description:Lysine 2-hydroxyisobutyrylation (Khib) is a novel naturally occurring PTM, which is firstly discovered in histone. The system Khib detection at proteomics level has been performed in various species and tissues for the function and role characterization of Khib in biological activities. However, the Khib study in plant species is relatively less and the plant root, one of the critical plant organ, haven’t been studied. In the present study, the first root tissues lysine 2-hydroxyisobutyrylome analysis was performed in wheat specie with antibody immunoprecipitation affinity and high resolution mass spectrometry-based proteomics. Bioinformatics analyses including function classification, subcellular location predication, gene ontology enrichment and pathway enrichment were conducted to demonstrate the roles of these Khib sites and proteins in wheat root. In total, 6328 Khib sites corresponding to 2186 proteins were repeatedly identified in three replicates. These Khib proteins showed a wide subcellular location distribution while cytoplasm was the primary subcellular compartment where Khib protein located. Function and pathways characterization of these Khib proteins indicated that many cellular functions and metabolism pathways potentially were affected by this modification. Protein and amino acid metabolism related process may be regulated by Khib, especially ribosome activities and proteins biosynthesis process. Carbohydrate metabolism and energy production related processes including glycolysis/gluconeogenesis, TCA cycle and oxidative phosphorylation pathways were also the hotspots where protein Khib occurred. Our work illustrated the potential regulation role of Khib in wheat leaf physiology and biology, which could be used as a useful reference for Khib studies in plant leaf.
Project description:Magnaporthe oryzae snodprot1 homologous protein (MSP1) has been shown to act as a pathogen-associated molecular pattern (PAMPs) and trigger PAMP-triggered immunity (PTI) response involving programmed cell death and expression of various defense-related genes in rice. The involvement of several post-translational modifications (PTMs) in the regulation of plant immune response, especially PTI, during pathogen infection is well established, however, the information on the regulatory roles of these PTMs in response to MSP1-induced signaling in rice is currently elusive. Here, we report the phosphoproteome, ubiquitinome, and acetylproteome to investigate the MSP1-induced PTMs alterations in MSP1 overexpressed rice. Our analysis identified a total of 4,666 PTM modified sites in rice leaves including 4,292 phosphosites, 189 ubiquitin sites, and 185 acetylation sites. Among these, PTM status of 437 phosphorylated, 53 ubiquitinated, and 68 acetylated peptides were significantly changed by MSP1. Functional annotation of MSP1 modulated peptides by MapMan analysis revealed that these were majorly associated with cellular immune responses such as signaling, transcription factors, DNA and RNA regulation, and protein metabolism, among others. Taken together, this study uncovers the MSP1-induced PTMs changes in rice proteins and identified several novel components of rice-MSP1 interaction.
Project description:Clinically translatable large animal models have become indispensable for cardiovascular research, clinically relevant proof of concept studies and for novel diagnostic and therapeutic interventions. In particular, the pig as emerged as an essential cardiovascular disease model, because its heart, circulatory system, and blood supply are anatomically and functionally similar to that of humans. Unfortunately, molecular and omics-based studies in the pig are hampered by the incompleteness of the genome and the lack of diversity of the corresponding transcriptome annotation. Here, we employed Nanopore long-read sequencing and in-depth proteomics on top of Illumina RNA-seq to enhance the pig cardiac transcriptome annotation. We assembled 15,926 transcripts, stratified into coding and non-coding, and validated our results by complementary mass spectrometry. A manual review of several gene loci, which are associated with cardiac function, corroborated the utility of our enhanced annotation. All our data are available for download and is also provided as tracks for integration in genome browsers. We deem this resource as highly valuable for molecular research in an increasingly relevant large animal model.
Project description:A differential label-fee proteomics approach was performed using 27 biopsies from patients with HCV-associated hepatic fibrosis. For statistical analysis the patients were grouped into a low and a high fibrosis group. The low fibrosis group contained 13 patients of fibrosis stages 0, 1 and 2, whereas the high fibrosis group contained 14 patients of fibrosis stages 3 and 4 (fibrosis stages according to Batts-Ludwig classification).
Project description:We have analysed a patinet derived mesothelioma cell line by immuniopeptidomics and identified cancer antignes in this line immunopeptidome.
Project description:Peptide vaccination remains a viable approach to induce T-cell mediated killing of tumours. To identify potential T-cell targets for Triple-Negative Breast Cancer (TNBC) vaccination, we examined the effect of the pro-inflammatory cytokine interferon-γ (IFNγ) on the transcriptome, proteome and immunopeptidome of the TNBC cell line MDA-MB-231. Using high resolution mass spectrometry, we identified a total of 84,131 peptides from 9,647 source proteins presented by human leukocyte antigen (HLA)-I and HLA-II alleles. Treatment with IFNγ resulted in a remarkable remoulding of the immunopeptidome, with only a 34% overlap between untreated and treated cells across the HLA-I immunopeptidome, and expression of HLA-II only on treated cells. IFNγ increased the overall number, diversity and abundance of the immunopeptidome, as well as the proportion of coverage of source antigens. The suite of peptides displayed under conditions of IFNγ treatment included many known tumour associated antigens, with the HLA-II repertoire sampling 265 breast cancer associated antigens absent from those sampled by HLA-I. Quantitative analysis of the transcriptome (10,248 transcripts) and proteome (6783 proteins) of these cells revealed 229 proteins and transcripts were commonly differentially expressed, most of which involved in downstream targets of IFNγ signalling including components of the antigen processing machinery such as tapasin and HLA. However, these changes in protein expression did not explain the dramatic modulation of the immunopeptidome following IFNγ treatment. These results demonstrate the high degree of plasticity in the immunopeptidome TNBC cells following cytokine stimulation and provide evidence that under pro-inflammatory conditions a greater variety of HLA-I and HLA-II vaccine targets are unveiled to the immune system. This has important implications for the development of personalised cancer vaccination strategies.
Project description:Aims/hypothesis AGEs are considered environmental contributors of type 1 diabetes, but the exact role AGEs play early in pathogenesis of the disease, remains unidentified. We aimed to reduce AGEs with the pharmacotherapy alagebrium chloride in MIN6N8 cells and early in life in NOD mice to determine its impact on beta cell immunogenicity, function, and disease progression. Methods MIN6N8 cells were cultured with AGEs with or without short-term alagebrium therapy to determine the effect on ER stress and beta cell antigen presentation via a reporter assay for protein responses in the unfolded protein response, the enzymatic activity of endoplasmic reticulum aminopeptidase-1 (ERAP1) and the immunopeptidome. To determine the effect of short-term alagebrium therapy on beta cells in vivo, female NOD mice were treated with alagebrium and insulin secretion, insulitis, and immune cell repertoire were studied. Adoptive transfer studies and diabetes progression studies were used to consider the impact of alagebrium on immune cell function and disease outcome. Results In MIN6N8 cells, alagebrium therapy inhibited the induction of endoplasmic reticulum (ER) stress and the activity of ERAP1 by AGE-modified proteins. Alagebrium treated-MIN6N8 cells did not change the MHC Class I presentation of known beta cell antigens but did induce proteomic changes related to ER homeostatic pathways. Prior to overt autoimmune diabetes, female NOD mice treated with alagebrium for 30-40 days had improved insulin secretion, reduced insulitis and amplified proportions of pancreatic CD8+ T cells, mature B cells and F4/80+ macrophages. Splenocytes from alagebrium-treated mice adoptively transferred disease to NODscid recipients and maintained interferon production in vitro. While partial protection of islets by alagebrium was seen following the adoptive transfer of activated NOD G9C8 transgenic TCR CD8+ T cells, alagebrium therapy in NOD mice did not reduce diabetes progression. Conclusions/interpretation Our data suggests that in experimental models of diabetes, short-term alagebrium treatment allows for beta cell improvement, and maintenance of immune cell function, which does not fully mitigate diabetes progression.
Project description:We found that USF2 is repressor of lysosomal genes. To find the interacting co-repressor complex of USF2, we performed USF2 complex purification and mass spectrography.
Project description:Roothans et al., analyzed heterotrophic denitrification processes that can be an important source of nitrous oxide. We employed planktonic nitrification-inhibited denitrifying enrichment cultures under alternating oxic-anoxic conditions. The dynamic conditions resulted in a general presence of the denitrifying enzymes. Overall, we show that aerobic denitrification should not be neglected as an ecologically relevant process. Contact author: m.laureni@tudelft.nl
Project description:A quantitative label-free secretome analysis protocol using a click chemistry-based approach for the enrichment of secreted glycoproteins was adapted for and applied to a T cell model. There, Jurkat cells were activated via PMA/ionomycin and the dynamic modulation of the T cell secretome was investigated and compared to the dynamic modulation of the T cell proteome of the same model.