Affinity-enrichment mass spectrometry profiling to determine the interacting partner of ginsentide TP1
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Prof James P. Tam
PROVIDER: PXD039905 | JPOST Repository | Wed Feb 07 00:00:00 GMT 2024
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 160226_CRPIP_Blank-01_01_A.raw | Raw | |||
| 160226_CRPIP_Blank-01_01_B.raw | Raw | |||
| 160226_CRPIP_Blank-01_02_A.raw | Raw | |||
| 160226_CRPIP_Blank-01_02_B.raw | Raw | |||
| 160226_CRPIP_Blank-02_01_A.raw | Raw |
Items per page: 5 1 - 5 of 20 |
Similar Datasets
Project description:Defining protein-protein interactions (PPIs) is central to the biological sciences. Here, we present a novel platform - Affinity Capture of Polyribosomes followed by RNA sequencing (ACAPseq) - for identifying PPIs. ACAPseq harnesses the power of massively parallel RNA sequencing (RNAseq) to quantify the enrichment of polyribosomes based on the affinity of their associated nascent polypeptides for an immobilized protein “bait”. This method was developed and tested using neonatal mouse brain polyribosomes and a variety of extracellular domains as baits. Of 92 baits tested, 25 identified one or more binding partners that appear to be biologically relevant; additional candidate partners remain to be validated. ACAPseq can detect binding to targets that are present at less than 1 part in 100,000 in the starting polyribosome preparation. One of the observed PPIs wes analyzed in detail, revealing the mode of homophilic binding for Protocadherin-9 (PCDH9), a non-clustered Protocadherin family member.
2018-10-20 | GSE121524 | GEO
Project description:We generated a new recombinant protein HARD (High-affinity RNA-binding Domain) with non-sequence-specific high-affinity on RNAs and developed the immobilized-HARD protein-mediated RNA affinity purification approach (HARD-AP).We showed that HARD-AP successfully captured all RNA biotypes.
2024-08-23 | GSE214173 | GEO
Project description:Proteins function in crowded cellular environments in which they must bind to specific target proteins but also avoid binding to many other off-target proteins. In large protein families this task is particularly challenging because many off-target proteins have very similar structures. How this specificity of physical protein-protein interactions in cellular networks is encoded and evolves is not very well understood. Here we address the question of specificity-encoding by comprehensively quantifying the effects of all mutations in one protein, JUN, on its binding to all other members of a protein family, the 54 human basic leucine zipper transcription factors. Fitting a global thermodynamic model to the data reveals that most affinity changing mutations equally affect JUN’s propensity to bind to all its interaction partners. Mutations that alter the specificity of binding are much rarer. These specificity-altering mutations are, however, distributed throughout the JUN interaction interface. JUN’s interaction specificity is encoded by both positive determinants that promote on-target interactions and negative determinants that prevent off-target interactions. Indeed, about half of the specificity-defining residues in JUN have dual functions and both promote on-target binding and prevent off-target binding. Whereas nearly all mutations that alter specificity are pleiotropic and also alter the affinity of binding to all interaction partners, the converse is not true with mutations outside of the interface able to tune affinity without affecting specificity. Our results provide the first global view of how mutations in a protein affect binding to all its potential interaction partners and reveal the distributed encoding of specificity and affinity in an interaction interface. They also show how the modular architecture of coiled-coils provides an elegant solution to the challenge of optimising specificity and affinity in a large protein family.
2024-05-02 | GSE245326 | GEO
Project description:Affinity enrichment mass spectrometry experiment to identify protein-protein interactions of hCOQ4-FLAG WT and hCOQ4-FLAG D164A.
2023-10-26 | MSV000093201 | MassIVE
Project description:Protein-protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein-protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers.
| S-EPMC4288248 | biostudies-literature
Project description:Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes and hence are of high interest for supporting wound healing. Although sulfated GAGs (sGAGs) are internalized into cells, knowledge of intracellular effects and interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins from human bone marrow stromal cells (hBMSC). Overall, 478 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. Only a limited fraction of about 10 % was annotated for extracellular regions. The interactions of the 2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and Serine protease HTRA1 (HTRA1) were confirmed in reverse assays.
2021-11-03 | PXD023362 | Pride
Project description:present study, we evaluated four small molecule affinity-based probes based on agarose-immobilized benzamidine (ABA), O-Phospho-L-Tyrosine (pTYR), 8-Amino-hexyl-cAMP (cAMP), or 8-Amino-hexyl-ATP (ATP) for their ability to remove high-abundant proteins such as serum albumin from plasma samples thereby enabling the detection of medium-to-low abundant proteins in plasma samples by mass spectrometry-based proteomics. We compared their performance with the commonly used immunodepletion method, the Multi Affinity Removal System Human 14 (MARS14) targeting the top 14 most abundant plasma proteins and also the ProteoMiner protein equalization method by label-free quantitative LC-MSMS analysis. The affinity-based probes demonstrated high reproducibility for low abundant plasma proteins, down to picomol per ml levels, compared to the MARS14 and the Proteominer methods, and also demonstrated a superior removal of the majority of the high abundant plasma proteins. The ABA-based affinity probe and the Proteominer protein equalization method performed superior compared to all other methods in terms of the number of analyzed proteins. All the tested methods were highly reproducible for both high-abundant plasma proteins and low abundant proteins as measured by correlation analysis of six replicate experiments. In conclusion, our results demonstrated that small-molecule based affinity-based probes are excellent alternatives to the commonly used immune-depletion methods for proteomic biomarker discovery studies in plasma.
2020-08-27 | PXD020727 | Pride
Project description:We identified a nuclear interactome of FMRP by combining subcellular fractionation of rat brains at the post-natal day 14 with pull down affinity purification and mass spectrometry analysis. By this approach, we have listed 55 candidate nuclear partners.
2022-08-14 | PXD034157 | Pride
Project description:Potential miR-202 targets were identified using a targetome-wide RIP-based microarray. HeLa cells were transfected with either a miR-202 mimic or a scrambled single-stranded RNA negative control. RNA was isolated from total cell lysate prior to IP and from antibody-immobilized Protein G agarose beads-RNP complexes (post-IP).
2012-12-19 | GSE42981 | GEO
Project description:Effector proteins are bacterial virulence factors secreted directly into host cells and, through extensive interactions with host proteins, rewire host signaling pathways to the advantage of the pathogen. Despite the crucial role of globular domains as mediators of protein-protein interactions (PPIs), previous structural studies of bacterial effectors are primarily focused on individual domains, rather than domain-mediated PPIs, which limits their ability to uncover systems-level molecular recognition principles governing host-bacteria interactions. Here, we took an interaction-centric approach and systematically examined the potential of structural components within bacterial proteins to engage in or target eukaryote-specific domain-domain interactions (DDIs). Our results indicate that: 1) effectors are about six times as likely as non-effectors to contain host-like domains that mediate DDIs exclusively in eukaryotes; 2) the average domain in effectors is about seven times as likely as that in non-effectors to co-occur with DDI partners in eukaryotes rather than in bacteria; and 3) effectors are about nine times as likely as non-effectors to contain bacteria-exclusive domains that target host domains mediating DDIs exclusively in eukaryotes. Moreover, in the absence of host-like domains or among pathogen proteins without domain assignment, effectors harbor a higher variety and density of short linear motifs targeting host domains that mediate DDIs exclusively in eukaryotes. Our study lends novel quantitative insight into the structural basis of effector-induced perturbation of host-endogenous PPIs and may aid in the design of selective inhibitors of host-pathogen interactions.
| S-EPMC8126662 | biostudies-literature