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:This project contains raw data, intermediate files and results used to create the PRIDE human phosphoproteome map. The map is based on joint reanalysis of 110 publicly available human datasets. All relevant datasets were retrieved from the PRIDE database, and after manual curation, only assays that employed dedicated phospho-enrichment sample preparation strategies (e. g. metal oxide affinity chromatography, anti-P-Tyr antibodies, etc.) were included. Raw files were jointly processed with MaxQuant computational platform using standard settings (see Data Processing Protocol). In total, the joint analysis allowed identification of 252,189 phosphosites at 1% peptide spectrum match false discovery rate (PSM FDR) (MQ search results available in ‘txt-100PTM’ folder), of which 121,896 passed the additional 1% site localization FDR threshold (MQ search results available in ‘txt-001PTM’ folder).
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.
Project description:Introduction: Cardiac myosin binding protein-C (cMyBP-C) becomes dephosphorylated in the failing heart and reduced phosphorylation-dependent regulation of cMyBP-C has been implicated in contractile dysfunction. To date, several phosphorylation sites have been identified for human cMyBP-C; however, a comprehensive characterization of the cMyBP-C phosphoproteome is lacking. This study aimed to characterize the cMyBP-C phosphoproteome using two different proteomic-based methods in explanted control and end-stage failing hearts. Methods: The first approach used to characterize the cMyBP-C phosphoproteome employed a strong-cation exchange chromatography (SCX)-based fractionation method (10 pooled samples, technical replicates = 4) and the second employed a sodium dodecylsulfate polyacrylamide gel electrophoresis method (n = 10; technical replicates = 2). Each subsequently underwent titanium dioxide (TiO2) affinity chromatography to enrich for the tryptic phosphopeptides, which were analyzed using an LTQ-Orbitrap mass spectrometer. Moreover, recombinant C0-C2 fragment of mouse cMyBP-C incubated with PKA, PKC, CamKII and CK2 was analyzed to identify the kinases involved with phosphorylation of cMyBP-C. Results: Seventeen phosphorylation sites on cMyBP-C were identified, with the majority localized in the N-terminal domains C0-C2. The three most abundant phosphorylated sites, Ser284, Ser286 and Thr290, are located in the regulatory M-domain of cMyBP-C. Ser284 showed a significant reduction in phosphorylation in HF, while Ser286 and Thr290 showed a trend towards a reduced phosphorylation. Conclusion: This study demonstrates that cMyBP-C is more extensively phosphorylated than previously known, with 10 novel sites identified. Most sites were primarily located within the N-terminal side of the protein. The three most highly phosphorylated sites on cMyBP-C were Ser284, Ser286 and Thr290 and these three sites showed decreased phosphorylation in the failing heart, which implicate their importance for fine-tuning contractility. To date, the functional importance of Ser286 and Thr290 is unknown. In addition, 16 sites were identified after in vitro kinase incubation. Bioinformatics pipeline: All RAW files were searched using the Sorcerer 2 Sequest algorithm. Human samples were searched against the IPI human database version 3.79 and mouse samples were searched against the IPI mouse database version 3.80. All searches were carried out with static modification of +57 on C and differential modifications of +16 on M and +80 on S,T,Y. Parent mass tolerance was set to 50 ppm and fragment mass tolerance was set to 1 Da. Post-search analysis was performed using Scaffold version 3.2.0 and site localization was determined using Scaffold PTM version 1.1.2 (Proteome Software, Inc., Portland, OR, USA).
Project description:Plasmodium falciparum schizont proteins were extracted using SDS, digested with trypsin and phosphopeptides were enriched using IMAC. Phosphopeptides were analysed using either decision tree or data dependent neutral loss triggered ETD acquisition. All raw MS data files were processed and converted into MGF file format using Proteome Discoverer 1.1 (Thermo Scientific). A precursor filter of 600-10000 Da and a non-fragment filter were applied to ETD spectra to remove un-reacted precursor peaks, charge reduced precursor peaks, neutral losses from charge reduced precursors and FT Overtones using default settings. All ion trap spectra with less than 15 fragmentation peaks were removed and a signal to noise filter of 3 was applied to all spectra. All datasets were searched using Mascot v2.2 (Matrix Science) against a combined Human (IPI, 2010) and Plasmodium falciparum (GeneDB) sequence database (79,637 sequences) using the following search parameters: trypsin with a maximum of 3 missed cleavages, 10 ppm for MS mass tolerance, 0.5 Da for MS/MS mass tolerance, with Acetyl (Protein N-term), Oxidation (M), Deamidated (NQ), Carbamidomethyl (C) and Phospho ST set as variable modifications. ETD spectra were searched using c, z and y ion series and CID data was searched using b and y ion series. All searches used Mascot’s automated decoy database searching.