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:Gene expression profiling of upland cotton line Im216 to inoculation with Xanthomonas campestris pv. malvacearum race 1. Fifth or sixth leaves of the bacterial blight-resistant cotton line Im216, which had been grown in a plant growth chamber, were infiltrated with a suspension of about 5x10^6 colony-forming units ml^-1 of Xanthomonas campestris pv. malvacearum race 1 in sterile saturated CaCO3 solution or were not inoculated (control). Keywords: Time-course Samples consisting of one leaf were harvested at 8, 14, 20, 30, 45, and 60 hpi. Three biological replicate experiments were performed on separate occasions. Total RNA was isolated, and from each sample 50 µg was reverse-transcribed along with non-plant normalizing Sp4 RNA (0.5 ng) and Sp5 RNA (0.05 ng), hybridized to slides for approximately 18 h, and detected with Cy5 (inoculated) or with Cy3 (non-inoculated) labeled dendrimers (hybridization for 3 h), using the 3DNA Array 350TM kit (Genisphere). Hybridization signals were measured using a ScanArray Express (PerkinElmer), and images were processed with GenePix Pro version 4.0 (Axon Instruments). Normalized log ratio VALUES were determined using the R-project statistical environment (<http://www.r-project.org/>), Bioconductor (<http://www.bioconductor.org/>) and the LIMMA package (Smyth 2004) through the GenePix AutoProcessor (GPAP, http://darwin.biochem.okstate.edu/gpap/>) website (H. Weng and P. Ayoubi, unpublished).
Project description:A prediction of peritoneal recurrence is of significance using metastasis-related biomarker. This work describes a combined analysis of proteome and transcriptome data for biomarker discovery in highly metastatic cell line. We used nano-flow liquid chromatography (LC) linear ion trap time-of-flight mass spectrometry (LIT-TOF MS) and cDNA microarray to identify specific protein differentially expressed between a highly metastatic stomach cancer cell line MKN-45-P and its parental cell line MKN-45. In total, 240 proteins were found to be expressed between the two cell lines. Of these, 75 proteins (31%) and 49 proteins (20%) were only identified from MKN-45-P and MKN-45 respectively. An mRNA expression of 1533 genes was up-regulated in MKN-45-P compared with MKN-45. No close correlation was found between proteomic and transcriptomic analysis. Interestingly, 4 of 240 proteins (1.6%) were involved in the up-regulated mRNA expression.
Project description:We have developed a multi-analyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular barcode. After staining a sample, T7 polymerase amplifies the tags which are then hybridized to a DNA microarray for indirect measurement of each analyte. Here we coupled 44 of the tags to aliquots of an IgG1 isotype negative control antibody and the 4 remaining tags we coupled to anti-CD3, anti-CD4, anti-CD19, and anti-CD20 to create a 48-plex HIT cocktail. We then used this cocktail to stain 1 x 10^6 T or B cells and during amplification incorporated either cyanine 3-UTP (Cy3-UTP) or Cy5-UTP. Additionally, we snap froze and thawed an aliquot of the cocktail (FT). Keywords: protein profiling, cell type comparison Two-condition experiment, T cells versus B cells, including dye-swaps and self-self experiments. We also tested an array that had been frozen and thawed.
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:A prediction of peritoneal recurrence is of significance using metastasis-related biomarker. This work describes a combined analysis of proteome and transcriptome data for biomarker discovery in highly metastatic cell line. We used nano-flow liquid chromatography (LC) linear ion trap time-of-flight mass spectrometry (LIT-TOF MS) and cDNA microarray to identify specific protein differentially expressed between a highly metastatic stomach cancer cell line MKN-45-P and its parental cell line MKN-45. In total, 240 proteins were found to be expressed between the two cell lines. Of these, 75 proteins (31%) and 49 proteins (20%) were only identified from MKN-45-P and MKN-45 respectively. An mRNA expression of 1533 genes was up-regulated in MKN-45-P compared with MKN-45. No close correlation was found between proteomic and transcriptomic analysis. Interestingly, 4 of 240 proteins (1.6%) were involved in the up-regulated mRNA expression. Comparison analysis of gene expression between highly metastatic gastric cancer cell line MKN-45-P and its parental cell line MKN-45.
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:Mitochondria are essential cellular organelles that participate in important cellular processes, including bioenergetics, metabolism, and signaling. Recent functional and proteomic studies have revealed the remarkable complexity of mitochondrial protein organization. Protein machineries with diverse functions such as protein translocation, respiration, metabolite transport, protein quality control and the control of membrane architecture interact with each other in dynamic networks. The goal of this study was to identify protein expression changes in a human cardiomyocyte cell line treated with several mitochondrial toxicants which inhibit mitochondrial membrane potential (MMP) and mitochondrial respiration. AC16 human cardiomyocyte cells were treated with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), dinoterb, picoxystrobin, pinacyanol and triclocarban for 18 h around the IC50 values generated from MMP assay. The samples were harvested and labeled with tandem mass tags with different mass isotopes. Peptide assignment was performed in Proteome Discoverer. Each dataset was analyzed in Ingenuity Pathway Analysis (IPA). In the proteomic profile, these compounds showed dysregulation of a group of mitochondrial proteins (e.g. NDUA, NDUB, BCS1, CYB5B and SDHF2), as well as proteins involved in lipid metabolism (e.g. CPT, MECR, and LPGAT1), cytoskeleton protein changes (e.g. CROCC, LAMC3, FBLN1 and FMN2) and stress response (e.g. IKBKG, IKBB, SYVN1, SOD2 and CPIN1). Proteomic data from the current study provides key insights into chemical induced cellular pathway dysregulation, supporting the use of proteomic profiling as a sensitive method to further explore molecular functions and disease pathogenesis upon exposure to environmental chemicals.
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.