Project description:Affinity purification-mass spectrometry (AP-MS) is a powerful approach for protein-protein interaction (PPI) analysis. Here, we report the development of a micro-scale immobilized metal affinity chromatography (m-IMAC) platform for rapid and reproducible purification of protein complexes prior to mass spectrometric analysis. The method was applied for mapping PPIs of Myc protein which regulates the expression of thousands of genes and plays a central role in tumorigenesis. Systematic characterization of Myc interactome is essential for understanding how a single transcription factor orchestrates such diverse biological processes. Affinity purification was performed using m-IMAC columns fabricated by photoinitiated polymerization within pipette tips (spin-tip columns), enabling efficient enrichment of Myc interacting proteins. Analysis with Label-Free Quantification (LFQ) with Data-Independent Acquisition (DIA) proteomics identified 574 Myc interacting proteins, the majority of which have not been previously reported as the Myc interactors. Two newly identified Myc associated factors were validated by biochemical assays, and the overall dataset showed good concordance with the reported Myc interactors. Gene Ontology (GO) enrichment analysis of the Myc associated proteome revealed significant enrichment in macromolecular metabolism (particularly RNA processing), regulation of gene expression, chromatin organization and remodeling. This work provides new insights into Myc mediated transcriptional regulation in proteomic level. Importantly, owing to its straightforward, rapid operation, the m-IMAC platform provides a general and efficient analytical strategy for high-throughput interactome analysis.
Project description:In this project, we used affinity purification and label-free quantitative proteomics to identify BMAL1-interacting proteinsin HEK293T cells.
Project description:To understand the functional roles of YTHDF1 in cellular senescence and aging, we lead the RNA sequence in WT or Ythdf1 deficiency colon epithelial cells. The results showed that the cholesterol biosynthesis related genes were upregulated in Ythdf1 KO mice. We also purified FLAG-YTHDF1 protein in HEK293T cells and did protein mass spectrometry, and found that the mTORC1 and TSC complex constituents mTOR, RPTOR, and TSC1/2 were identified in the YTHDF1-complex. Methylated RNA immunoprecipitation(MeRIP) with specific m6A antibody and used for library construction and the next generation sequencing, to identify m6A modified transcripts in WT or Ythdf1 deficiency colon epithelial cells after DSS treatment
Project description:Antibody-based affinity purification of macromolecular complexes has revolutionized the study of protein-protein interactions. Here, we present AptA-MS (Aptamer Affinity – Mass Spectrometry), a robust strategy using a specific, high-affinity RNA aptamer against GFP to identify novel interactors of a GFP-tagged protein using high resolution MS. AptA-MS offers a high signal-to-noise ratio due to the absence of immunoprecipitation-derived contaminants and allows the identification of post-translational modifications without the need for modification-specific enrichments.
Project description:MS2-affinity purification coupled with RNA sequencing (MAPS) reveals S. aureus RsaG sRNA targetome. Affinity purification of in vivo regulatory complexes coupled with high throughput RNA sequencing methodology or MAPS standing for “MS2 affinity purification coupled to RNA".
Project description:To systematically map the arenavirus‒host protein interaction, we employed a systematic affinity purification mass spectrometry (AP-MS) approach to define the human proteins that physically interact with LCMV proteins. The four LCMV proteins (NP, LP, GPC, Z) and control protein GFP were tandemly cloned with Twin-Strep tags at C-terminal based on the same backbone pCAGGs vector respectively. Proteins were expressed in HEK293T cells followed by affinity-purification based on Strep-Tactin System. We summarized the number of human proteins interacting with each indicated LCMV protein baits and identified 284 human proteins with high-confidence PPIs (log2 enriched fold > 1, P < 0.05).
Project description:We sought to compare differences in the SARS-CoV-2 virus-human protein interaction networks between mutated (from the SARS-CoV-2 variants of concern) and wild-type viral proteins. Here, we ectopically expressed 2x-strep-tagged constructs encoding mutant or wild-type SARS-CoV-2 viral proteins in HEK293T cells followed by affinity purification mass spectrometry (APMS). This allowed us to quantify differences in protein-protein interactions attributable to mutations present within SARS-CoV-2 variant proteins.
Project description:The aims of this project is to establish a comprehensive proteomic map of the lysosomes using isolated lysosomes via affinity purification. Affinity-purified mitochondria was used as a control to demonstrate the organelle specificity. In this study, we identified several lipid transporters that are associated with the lysosome and further characterized their roles in sterol-dependent mTORC1 regulation.
Project description:The goal of this project is to identify interacting proteins of Flag-tagged STING before and after cGAMP stimulation. STING knockout (STING-KO) BJ cells stably re-expressing Flag-STING were treated with 1 M cGAMP and whole cell lysates were harvested 30 min later for IP with anti-Flag M2 affinity gel. STING-KO cells without Flag-STING expression were used as a negative control.
Project description:In this study, we use DNA affinity purification sequencing to identiy genome-wide binding of LFY transcription factor, a master regulator of flower development in Arabidopsis. We generated two sets of data, one using genomic DNA from plant tissue, thus retain DNA methylation, as probe for DNA affinity purification (DAP-seq dataset), and the other using PCR amplified genomic DNA (without DNA methylation; AmpDAP-seq dataset).