TMT6plex quantitative proteomics of 293T NME4 KO cell
Ontology highlight
ABSTRACT: To identify the role NME4,we knockout NME4 in 293T cells. Followed by protein extraction, trypsin digestion, TMT6 labeled, HPLC fractionation and LC-MS/MS.
Project description:To identify interacting proteins of NME4 in human cell lines, we using tandem affinity purification followed by mass spectrometry (TAP-MS) in HEK293T cells. We established stable cell line which expressing NME4 gene fused with SFB triple tags (S tag-2x Flag tag-SBP tag) or TurboID. We isolated, combined, and affinity-purified the membrane and soluble fractions using TAP. We identified proteins associated with the bait in the isolated complexes using MS and searched the uniprot human database for these proteins.
Project description:To identify the proteins interact with PDL1 protein in human cell lines, we analyzed the protein complexes using tandem affinity purification followed by mass spectrometry (TAP-MS) in HEK293T. We established the cell lines stably expressing PDL1 gene fused with Flag tags. We identified proteins associated with the bait in the isolated complexes using MS and searched the human databases for these proteins.
Project description:To identify the proteins interact with LncRNA SNHG6 in human cell lines, we analyzed the protein using RNA-Pulldown followed by mass spectrometry in HEPG2. We identified proteins associated with the SNHG6 sense/anti-sense in the isolated complexes using MS and searched the human databases for these proteins.
Project description:There are several methods to bring down the bait and interactors from the cell lysates, each with its own advantages and disadvantages. Recently, the most commonly used method is one step AP, TAP and proximity-labeling. In order to analyze the Strengths and weaknesses of the three methods, fusion proteins were constructed by NICD4 with FLAG, SFB (S tag-2x Flag tag-SBP tag) and TurboID respectively, NICD4 and its interactors were obtained by purification. By analyzing these proteins, we believe that SFB-TAP is the most reliable and effective method to identify interacting proteins.
Project description:MS analysis of proteins interacting with PKA and TGF-β1 in HEK293T cells. MS analysis of immunoprecipitated TGF-β1 in HEK293T cells with TGF-β1 overexpression.
Project description:Using a systematic proteomic approach, we define the protein-protein interaction network for PLD superfamily and phosphatidic acid in human cells and reveal diverse cellular signaling events involved for this key protein family and lipid.
Project description:Aedes aegypti [Linnaeus in Hasselquist (Diptera: Culicidae); yellow fever mosquito] transmits several viruses that infect millions of people each year including, Zika, dengue, yellow fever, chikungunya, and West Nile. Disease transmission occurs during blood feeding. Only the females blood feed as they require a bloodmeal for oogenesis. In the bloodmeal, females receive a substantial iron load in the forms of holo-transferrin and hemoglobin. We are interested in the effects of the iron in a bloodmeal on the expression of proteins during oogenesis. Our previous data showed that during digestion of a blood meal, the gut iron concentration decreases 10-fold, while that of the ovaries doubles from ingestion to 72 hours post feeding. Approximately 72 hours post feeding, eggs are laid with ~125 ng Fe each. We are interested in the effects of the blood meal iron on the expression of proteins detected in the ovaries during the early oogenesis, 24 hours post feeding, before eggs are laid. We have used tandem mass tag-labeling proteomics to quantify proteins expressed at this early stage following feeding of a controlled iron diet. Our findings provide the first quantitative report of differential ovary protein expression in early oogenesis in mosquitoes fed three different iron diets.
Project description:As the remarkable prevalence of activated protein tyrosine kinases (TKs) as oncoproteins and their mutations being identified in numerous cancers, the control of protein tyrosine phosphorylation has been considered to play a central role in ensuring the homeostasis of cellular physiology and thus, preventing tumorigenesis. Protein tyrosine phosphatases (PTPs) can contribute to this equilibrium of protein tyrosine phosphorylation and thereby antagonize the oncogenic activities of tyrosine kinases, therefore, PTPs are prominently considered to act as tumor suppressors. To achieve a comprehensive understanding of the protein-protein interaction network for this PTP family, we isolated the ~ 70 PTP-associated protein complexes from HEK293T cells and provided a systematically proteomic analysis for this tumor suppressor family.