Mass Spectrometry of T47D cells treated with PTK6 PROTAC
Ontology highlight
ABSTRACT: PTK6 PROTAC treatment of T47D breast cancer cells were analyzed with mass spectrometry for determining PTK6 PROTAC specificity and global proteomic changes.
Project description:Retinoblastoma (RB) is an intraocular childhood tumor which, if left untreated, leads to blindness and mortality. Nucleolin (NCL) protein which is differentially expressed on the tumor cell surface, binds ligands and regulates carcinogenesis and angiogenesis. We found that NCL is over expressed in RB tumor tissues and cell lines compared to normal retina. We studied the effect of nucleolin-aptamer (NCL-APT) to reduce proliferation in RB tumor cells. Aptamer treatment on the RB cell lines (Y79 and WERI-Rb1) led to significant inhibition of cell proliferation. Locked nucleic acid (LNA) modified NCL-APT administered subcutaneously (s.c.) near tumor or intraperitoneally (i.p.) in Y79 xenografted nude mice resulted in 26 and 65% of tumor growth inhibition, respectively. Downregulation of inhibitor of apoptosis proteins, tumor miRNA-18a, altered serum cytokines, and serum miRNA-18a levels were observed upon NCL-APT treatment. Desorption electrospray ionization mass spectrometry (DESI MS)-based imaging of cell lines and tumor tissues revealed changes in phosphatidylcholines levels upon treatment. Thus, our study provides proof of concept illustrating NCL-APT-based targeted therapeutic strategy and use of DESI MS-based lipid imaging in monitoring therapeutic responses in RB.
Project description:Methanococcus maripaludis is a methanogenic archaeon. Within its genome, there are two operons for membrane associated hydrogenases, eha and ehb. To investigate the regulation of ehb on the cell, an S40 mutant was constructed in such a way that a portion of the ehb operon was replaced by pac cassette in the wild type parental strain S2 (done by Whitman's group at the University of Georgia). The S40 and S2 strains were grown in 14N and 15N media with acetate separately. A biological replicate was made by switching the media. Mass spectrometry based quantitative proteomics were done on the mixtures to investigate the differences in expression patterns between S40 and S2. Keywords: isotope labeling mass spectrometry, quantitative proteomics
Project description:Dinaciclib potently inhibits CDK12, resulting in decreased mRNA from many genes associated with DNA damage signaling and repair. We used microarrays to assess changes in global mRNA levels following dinaciclib treatment in breast cancer cells
Project description:Rice is one of the most important staple food and model species in plant biology, yet its quantitative proteomes are largely uncharacterized. Here we quantify the relative protein levels of over 15,000 genes across major rice tissues using a tandem mass tag strategy followed by intensive fractionation and mass spectrometry. We identify tissue-specific and -enriched proteins that are linked to the functional specificity of individual tissues. Proteogenomic comparison of rice and Arabidopsis reveals conserved proteome expression, which differs from mammals in that there is a strong separation of species rather than tissues. Notably, profiling of N6-methyladenosine (m6A) across the rice major tissues shows that m6A at untranslated regions is negatively correlated with protein abundance and contributes to the discordance between RNA and protein levels. We also demonstrate that our data are valuable for identifying novel genes required for regulating m6A methylation. Taken together, this study provides a paradigm for further research into rice proteogenome.
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