Project description:Gene expression profiles of RNA extracted at 24 or 48h from End1 cells infected with Chlamydia trachomatis or uninfected controls. This experiment forms part of the analysis of phosphoproteome changes after C.trachomatis infection.
Project description:Shotgun phosphoproteome of Sulfolobus acidocaldarius using PAciFIC technique. Briefly, proteins were denatured, alkylated, trypsine digestion, SCX separation Fractionated peptides were analysed on Bruker HCTultra. Data processing and bioinformatics: data from mass spectrometry were then extracted to mgf format using Bruker Data Analysis V4.0 with a MRM script, these were then were searched against the Sulfolobus acidocaldarius database (containing 2223 proteins) downloaded from NCBI in March 2010 using Phenyx V 2.6 (Genebio, Geneva). The searches were performed using parameters as follows: carbamidomethylation of cysteine (fixed modification), oxidation of methionine (variation), and phosphorylation of serine, tyrosine, threonine (variation), trypsin with 2 missed cleavages. Furthermore, other parameters such as parent, MS/MS, tolerances were set at 2.0 and 0.8 Da, respectively, whilst minimum peptide length, z-score, p-value and AC score were set at 5, 5.5, 10-5, and 5.5 respectively.
Project description:We investigated the differential phosphoproteome of the mouse heart after isoproterenol stimulus of the AC3-I and AC3-C mice. The former is a model of specific in vivo CaMKII inhibition by a transgenically expressed peptide, whereas the latter is a transgenic mouse expressing a control peptide. Data processing: all raw data files of the individual SCX fractions of each of the 2 mouse experiments were imported into Proteome Discoverer v1.3.0.339 and the combined peak list was split into CID and HCD data (where applicable) before database searching. Subsequently, CID and HCD peak lists were searched individually against an International Protein Index (IPI; http://www.ebi.ac.uk/ipi) database containing mouse sequences and common contaminants such as bovine serum albumin and human keratins (IPI-Mouse v3.84; 60 248 sequences) through a direct connection to our in-house Mascot server (Mascot v2.3.2, Matrix Science, London, UK). The following settings were used: carbamidomethylation on cysteines as static modification; light, intermediate, and heavy dimethylation of peptide N-termini and lysine side chains, as well as oxidation on methionine and phosphorylation on serine, threonine, or tyrosine as variable modifications; and precursor mass tolerance of 20 ppm and 0.8 Da on the fragment masses (for CID) but 20 ppm and 0.02 Da for HCD searching. The enzyme was specified as trypsin, and 2 missed cleavages were allowed.
Project description:Phosphoinositide 3-kinase (PI3K) signaling activation is frequently observed in triple negative breast cancer, however, PI3K inhibitors have shown limited clinical activity. To investigate resistance mechanisms, we performed global transcriptome, proteome, phosphoproteome and kinome analysis of a panel of triple negative breast cancer patient derived xenograft models with varying responsiveness to buparlisib, a pan-PI3K inhibitor, for differentially expressed baseline and post-treatment biomarkers. Resistance was associated with incomplete inhibition of PI3K and upregulated MAPK/MEK signaling in response to buparlisib. Outlier phosphoproteome and kinome analyses identified additional candidates in association with buparlisib resistance, including NEK9 and MAP2K4. Knockdown of NEK9 or MAPK2K4 reduced both baseline and feedback MAPK/MEK signaling and enhanced buparlisib efficacy in vitro. Interestingly, we show that a complex ins/del in PIK3CA led to a change in buparlisib response in a NEK9/MAP2K4 dependent manner. In summary, our study indicates a role for NEK9 and MAP2K4 in mediating buparlisib resistance and demonstrates the value of unbiased global analyses in uncovering resistance mechanisms to targeted therapy.
Project description:MES cells were treated with vehicle or 250 nM extracellular alpha-synuclein and plasma membrane proteins were labeled with biotin, followed by affinity purification. The biotinylated enriched membranous proteins were incubated with an alpha-synuclein peptide array membrane, washed extensively with TBS-T, followed by incubation with avidin-HRP. The positive spots were excised and the bound proteins were eluted. The elutions from consecutive spots were combined before trypsin digestion and analysis of peptides by LC-MS/MS.