Project description:NAA10-mediated N-terminal acetylation is widespread and has been considered essential for viability in many model organisms. However, a Naa10 null mouse model is viable and has intact global N-terminal acetylation levels. The purpose of this project was to assess the N-terminal acetyltransferase activity of NAA15 immunoprecipitates from Naa10-KO mouse liver. We show that there is a novel paralog of Naa10 present in mice, termed Naa12, with a NatA-type enzymatic activity. We demonstrate the presence of Naa12 by detecting unique Naa12 peptides in NAA15 immunoprecipitates.
Project description:We collected 14 stock Hela aliquots from 13 different laboratories across the globe and cultured them in the same conditions. We extensively profiled the genome-wide copy numbers, mRNAs, proteins, protein turnover rates by genomics techniques and the highly reproducible and accurate proteomic method, SWATH mass spectrometry. The cell lines were also phenotyped with respect to the ability of transfected Let7 mimics to modulate Salmonella infection. We discovered significant heterogeneity between Hela variants especially differences between the CCL2 and Kyoto lines collected from different sites. In addition, we observed progressive divergence within a specific cell line over 50 successive passages. By associating proteotype and phenotype we identified molecular patterns that varied between cell lines and explained varying responses to Salmonella infection across the cells. The results furthermore quantify how the cells respond to genomic variability across the transcriptome and proteome.
Project description:Calcium/calmodulin-dependent protein kinase II (CaMKII) was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. However, the specific functions of the CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury have not been investigated yet. Thus, we studied the roles of the CaMKII isoforms and splice variants in I/R by the use of various CaMKII mutant mice. CaMKIIδC was up-regulated already one day after I/R injury but surprisingly, acute I/R injury was neither affected in CaMKIIδ-deficient mice, CaMKIIδ-deficient mice in which the splice variants CaMKIIδB and C were re-expressed nor in conditional CaMKIIδ/γ double-knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction. Leukocyte infiltration was not altered one day but five days after I/R, explaining the late effects of CaMKII deletion on post-I/R remodeling. Other than reported before, we demonstrate that CaMKII is not critically involved in the immediate mechanisms that regulate acute I/R injury but in the process of post-infarct remodeling. We analysed 6 groups in total: 3 groups from wild-type control animals and 3 groups from CaMKII delta/gamma double-KO mice. Sham operated animals served as controls in both wild-type and KO animal groups. 2 different time points in ischia/reperfusion operated animals were investigated: 1 day and 5 days post-surgery.
Project description:Investigation of whole genome gene expression level changes in a Bacteroides fragilis NCTC 9343 delta-ungD1 delta-ungD2 delta-PSH triple mutant, compared to the wild-type strain. The mutations engineered into this strain render it acapsular. The mutants analyzed in this study are further described in Coyne, M. J., M. Chatzidaki-Livanis, L. C. Paoletti, and L. E. Comstock. 2008. Role of glycan synthesis in colonization of the mammalian gut by the bacterial symbiont Bacteroides fragilis. PNAS 105(35):13098-13103 (PID 18723678). A six chip study using total RNA recovered from three separate wild-type cultures of Bacteroides fragilis NCTC 9343 and three separate cultures of a triple mutant strain, Bacteroides fragilis NCTC 9343 delta-ungD1 delta-ungD2 delta-PSH, in which ungD1 (BF1706), ungD2 (BF2848), and six genes (BF3454 through BF3459) of the PSH capsular polysaccharide locus are truncated or deleted entirely. Each chip measures the expression level of 4,302 genes from Bacteroides fragilis NCTC 9343 and the associated plasmid pBF9343 with fourteen 24-mer probe pairs (PM/MM) per gene, with three-fold technical redundancy.
Project description:Investigation of the whole genome expression level changes in phosphate limited Bacillus subtilis wild-type and delta-phoPR cells Investigation of the whole genome expression level changes of wild-type and delta-phoPR Bacills subtilis cells comparing high and low phosphate medium For each sample analyzed in this study 3 technical replicates were performed. 3 different samples were taken for wild-type cell and delta-phoPR cell, respectively. Samples were taken from exponentially growing cells in high and low phosphate medium as well as from phosphate-limited cells.
Project description:Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS) is a DIA method whose use in proteomic studies has increased considerably within the past five years. SWATH-MS acquires a complete and permanent digital record for all the detectable MS/MS spectra of a sample using DIA. After their generation, the SWATH-MS maps can be used for iterative analyses of candidate proteins. As with any analytical methodology that has potential widespread use, several studies have been conducted to optimize and evaluate the performance of SWATH-MS. The present dataset was acquired from 103 tissue samples of high-grade serous ovarian carcinoma collected and processed in the context of the CPTAC initiative and analyzed via bottom-up SWATH mass spectrometry.
Project description:In living cells most proteins are organized in stable or transient functional assemblies known as protein complexes, which control a multitude of vital cellular processes such as cell cycle progression, metabolism, and signal transduction. System-wide workflows for analysis of protein complexes using mass spectrometry based proteomics consists of a biochemical fractionation methods which take advantage of biophysical features such as charge or mass of protein complexes. In this project, we apply size exclusion chromatography to investigate protein assemblies from a Jurkat whole cell lysate. We introduced a fast and robust in-plate sample preparation workflow which allows for replicates to be performed with fewer technical hurdles and with a high degree of reproducibility) We acquired the data in SWATH-mode in combination with a high-throughput LC-system (EVOSEP one). The bioinformatics pipeline builds on the OpenSWATH workflow combined with CCprofiler, a tool for statistical scoring of protein co-elution profiles. This method enabled us to identify more than 100 protein complexes from mammalian cells within one day of measurement.
Project description:Pseudomonas aeruginosa is a virulent opportunistic pathogen responsible for high morbity in COPD, burns , implanted medical devices and cystic fibrosis. Pseudomonas aeruginosa is a problematic colonizer of the human lung. P. aeruginosa produces a phospholipase C (PlcH) that degrades choline-containing lipids such as phosphatidylcholine and sphingomylein that are found in lung surfactant and in host membranes. In this study, we analyzed gene expression in mutants defective in PlcH production (delta-plcH and delta-gbdR) and the wild type when growing in medium with lung surfactant. Pseudomonas aeruginosa was cultured in liquid cultures with aeration in a defined medium with Survanta, a lung surfactant replacement. Cultures were harvested during mid-exponential phase, and RNA was isolated for microarray analysis. The P. aeruginosa strain PAO1 wild type gene expression was compared to expression profiles from delta-gbdR and delta-plcHR deletion mutants, two mutants defective in PlcH production.
Project description:Remodeling of the extracellular matrix (ECM) is a common feature in lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Here, we applied a sequential tissue extraction strategy to describe disease-specific remodeling of human lung tissue in disease, using end-stages of COPD and IPF. Our strategy was based on quantitative comparison of the disease proteomes, with specific focus on the matrisome, using data-independent acquisition and targeted data analysis (SWATH-MS). Our work provides an in-depth proteomic characterization of human lung tissue during impaired tissue remodeling. In addition, we show important quantitative and qualitative effects of the solubility of matrisome proteins. COPD was characterized by a disease-specific increase in ECM regulators, metalloproteinase inhibitor 3 (TIMP3) and matrix metalloproteinase 28 (MMP-28), whereas for IPF, impairment in cell adhesion proteins, such as collagen VI and laminins, was most prominent. For both diseases, we identified increased levels of proteins involved in the regulation of endopeptidase activity, with several proteins belonging to the serpin family. The established human lung quantitative proteome inventory and the construction of a tissue-specific protein assay library provides a resource for future quantitative proteomic analyses of human lung tissues.