Project description:To identify substrates of the ubiquitinating E3 enzyme Rsp5 we applied purified Rsp5 to duplicate protein arrays. The Rsp proteins were expressed as fusion proteins to GST. We used as a control Ubr1, a RING domain containing E3 ligase We analyzed Rsp5 from S.cerevisiae on duplicate arrays, with four control chips, two without Rsp5 and two with Ubr1.
Project description:This SuperSeries is composed of the following subset Series: GSE24037: Salivary cytokine alterations in HIV infection part 1 GSE24064: Salivary cytokine alterations in HIV infection part 2 Refer to individual Series
Project description:We report an integrated analysis incorporating DNA copy number analyses, somatic exon mutations, mRNA expression via RNA-sequencing, and shotgun mass spectrometry analysis of protein abundance in 108 surgically resected squamous cell lung cancers (SCC) with accompanying clinical outcome, evaluation of tumor pathology, and other clinically relevant data. We identified three major subtypes of SCC at the proteomic level, with two groups associated with inflammation/immune response or oxidation-reduction biology. Inflamed tumors could be further sub-classified based on neutrophil infiltration or antigen presentation proteomes and reflected patterns of infiltrating immune cells. No gene mutations, mRNA signatures, or proteomic subclasses were associated with outcomes; however, the presence of B-cell rich tertiary lymph node structures could be associated with better patient outcomes. By integrating our proteogenomic data with publicly available RNA interference screen data, we identified TP63, PSAT1, and AKR1C3 as vulnerabilities in SCC, particularly in the redox proteomic group. This cohort and its deep molecular data serves as an important resource to better understand biology and targets associated with SCC.
Project description:Multi-omic absolute quantitative analysis of the yeast (Saccharomyces cerevisiae) mitotic cell cycle. Transcriptomics (RNA-Seq), proteomics (SILAC/ iBAQ), phosphoproteomics (SILAC/ iBAQ, enrichment with TiO2), and untargeted metabolomics (Metabolon, Inc.) were performed, all in biological triplicate. Three sub-projects from this central project were generated, in order of priority: (1) growth on glucose (n= 30 samples) (2) growth on ethanol (n= 21) and (3) pheromone effect (n= 51 samples; combined glucose and ethanol samples). For each sample, every omic type was analysed (n=4; transcriptomic, proteomic, phosphoproteomic, and metabolomic). Total omic samples generated for project = 204.
Project description:Cell line-based proteomics studies are susceptible to intrinsic biological variation that contributes to increasing false positive claims; most of the methods that follow these changes offer a limited understanding of the biological system. We applied a quantitative proteomic strategy (iTRAQ) to detect intrinsic protein variation across SH-SY5Y cell culture replicates. More than 95% of the quantified proteins presented a coefficient of variation (CV) <20% between biological replicates and the variable proteins, which included cytoskeleton, cytoplasmic and housekeeping proteins, are widely reported in proteomic studies. We recommend this approach as an additional quality control before starting any proteomic experiment.
Project description:We determined the effect of p53 activation on de novo protein synthesis using quantitative proteomics of newly synthesized proteins (pulsed stable isotope labeling with amino acids in cell culture, pSILAC) in combination with mRNA and non-coding RNA expression analyses by next generation sequencing (RNA-, miR-Seq) in the colorectal cancer (CRC) cell line SW480. Furthermore, genome-wide DNA binding of p53 was analyzed by chromatin-immunoprecipitation (ChIP-Seq). Thereby, we identified differentially regulated mRNAs (1258 up, 415 down), miRNAs (111 up, 95 down), lncRNAs (270 up, 123 down) and proteins (542 up, 569 down). Changes in mRNA and protein expression levels showed a positive correlation (r = 0.50, p < 0.0001). More transcriptionally induced genes displayed occupied p53 binding sites (4.3% mRNAs, 7.2% miRNAs, 6.3% lncRNAs, 5.9% proteins) than repressed genes (2.4% mRNAs, 3.2% miRNAs, 0.8% lncRNAs, 1.9% proteins), suggesting indirect mechanisms of repression. Around 50% of the downregulated proteins displayed seed-matching sequences of p53-induced miRNAs in the corresponding 3’-UTRs. Moreover, proteins repressed by p53 significantly overlapped with those previously shown to be repressed by miR-34a. We confirmed upregulation of the novel direct p53 target genes LINC01021, MDFI, ST14 and miR-486 and showed that ectopic LINC01021 expression inhibited proliferation in SW480 cells. Furthermore, HMGB1, KLF12 and CIT mRNAs were confirmed as direct targets of the p53-induced miR-34a, miR-205 and miR-486-5p, respectively. In line with the loss of p53 function during tumor progression, elevated expression of HMGB1, KLF12 and CIT was detected in advanced stages of cancer. This study provides new insights and a comprehensive catalogue of p53-mediated regulations and p53 DNA binding in CRC cells.
Project description:Analog-sensitive kinase technology allows for specific inhibition of a given kinase. Here we utilize an analog-sensitive CDK9 cell line to determine the impact of CDK9 inhibition on the phospho-proteome of human cells. Specifically, we aim to identify substrates of CDK9 in a quantitative way using stable isotope labeling by amino acids in cell culture (SILAC) followed by LC-MS/MS.
Project description:The endothelium is the frontline target of multiple metabolic stressors and pharmacological agents. As a consequence, endothelial cells (ECs) display highly dynamic and diverse proteome profiles. We describe here the culture of human aortic ECs from healthy and type 2 diabetic donors, the treatment with a small molecular conformation of trans-resveratrol and hesperetin (tRES+HESP), followed by proteomic analysis of whole-cell lysate. A number of 3666 proteins were presented in all the samples and thus further analyzed. We found that 179 proteins had a significant difference between diabetic ECs vs. healthy ECs, while 81 proteins had a significant change upon the treatment of tRES+HESP in diabetic ECs. Among them, 16 proteins showed a difference between diabetic ECs and healthy ECs and the difference was reversed by the tRES+HESP treatment, with the top 5 drastically altered proteins being ACVRL1, ADAM9, ITGAV, PCCB, and TGFBR2. Follow-up functional assays identified ACVRL1 and TGFBR2 as the most pronounced mediator for tRES+HESP-induced protection of angiogenesis in vitro. Our study has revealed the global changes in proteins and biological pathways in ECs from diabetic donors, which are potentially reversible by the tRES+HESP formula. Furthermore, we have identified the TGFβ signaling axis as a responding mechanism in ECs treated with this formula, shedding light for future studies for deeper molecular characterization