Project description:Comparison of mucus proteins from 6 different segments (from stomach to distal colon). 6 biological replicates, 2 MS replicates. The mucus that protects the surface of the gastrointestinal tract is rich in specialized O-glycoproteins called mucins, but little is known about other mucus proteins or their variability along the tract. We combined collection of mucus from explant tissues with FASP processing and single-shot mass spectrometry in an LTQ-Orbitrap system, to characterize the proteome of the murine mucus from stomach to distal colon. We identified ~1,300 proteins in the mucus, and found no differences in the protein composition or abundance between genders, but clear differences in the different gastrointestinal locations. Qualitatively, there was a relatively stable core proteome (~80% of the total proteins identified). Quantitatively, we found significant differences (~40% of the proteins) that could reflect mucus specialization throughout the gastrointestinal tract. Hierarchical clustering pinpointed a number of proteins that correlated with the main intestinal mucin, Muc2 (e.g. Clca1, Zg16, Klk1). This study provides a deeper knowledge of the gastrointestinal mucus proteome that will be important in further understanding this poorly studied mucosal protection system. Bioinformatics pipeline: Data was acquired in a hybrid LTQ-Orbitrap XL instrument (Thermo Scientific) in dependent mode, measuring full MS in the Orbitrap and selecting the 8 most abundant multiply charged ions for collision (CID, 30% normalized collision energy) and acquisition in the LTQ. Full MS scans were performed in the m/z 350-2,000 range, with internal calibration by lock mass (m/z 371.1012, siloxane), and resolution of 60,000. MS/MS scans were set at a target value of 100,000, with isolation width of 3 amu. The raw files obtained were analyzed in the MaxQuant 1.2.2.5 environment. Data were searched with the Andromeda search engine integrated in MaxQuant against an in-house database containing all the mucin sequences available (http://www.medkem.gu.se/mucinbiology/databases/), the UniProt-SwissProt mouse database (version 1203, reviewed sequences), and the standard MaxQuant contaminant database. Oxidation of methionines and acetylation of the protein N-terminus were set as variable modifications, and carbamidomethylation of cysteines as fixed; enzyme cleavage rules were defined for trypsin/P, with a maximum of 2 missed cleavages. Tolerances were limited as maximum 5 modifications per peptide, 20 ppm error for the first search and 6 ppm for the main search. Isoleucine and leucine were considered indistinguishable. The false discovery rate was calculated from searches against a reversed database, and set to 0.01 for proteins, peptides and modified sites. The identification rate was improved by matching between runs through remapped retention time (window of 2 min). The resulting data were loaded as protein groups into Perseus 1.3.0.4. Protein quantities were calculated as intensity values (from the extracted ion current: XIC) and normalized in ppm to the total XIC for the standard labeled peptides (AQUA) in each run.

Project description:Isolated murine kidney glomeruli were lysed in buffer containing 8M urea, and proteins were reduced and alkylated. Proteins were digested with trypsin. After reverse-phase chomatrography, peptides were fractionated using strong cation exchange chromatography. Phosphopeptides were enriched using IMAC (FeNTA) columns. Peptides were analyzed by LC-MS/MS on a Q Exactive mass spectrometer or an LTQ Orbitrap XL mass spectrometer. Metadata for Orbitrap samples (MaxQuant Output files include „Orbi“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; tobias.lamkemeyer@uni-koeln.de b. Instrument manufacturer, model: LTQ-Orbitrap XL, Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 2 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Linear Trap. 3.2. Activation/Dissociation: CID 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 60 seconds and followed by the acquisition of 5 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used. Metadata for QExactive samples (Max Quant output files include „QE“) 1. General features – 1.1 Global descriptors a. Responsible person or role: Markus Rinschen. markus.rinschen@uk-koeln.de; Marcus Krüger marcus.krueger@mpi-bn.mpg.de b. Instrument manufacturer, model: Q Exactive Thermo Scientific c. customization 2. Ion sources – ESI fed by nLC 1000 (Proxeon) 3. Post source component 3.1. Analyser: MS1 survey scan in an Orbitrap and MS2 analysed in Orbitrap/HCD cell 3.2. Activation/Dissociation: HCD 4. Spectrum and peak list generation and annotation 4.1. Data acquisition: MaxQuant v. 1.3.05 Top one method with a cycle of one full MS1 scan in the Orbitrap, followed by the fragmentation with dynamic exclusion window of 20 seconds in the HCD cell and followed by the acquisition of 10 product ion scans generated in the LTQ analyser, and detected in the LTQ. Unselected fragmentation. For further details, see .raw files. URL of file: Filename 4.2. Data analysis: MaxQuant v 1.3.05 Parameters used in the generation of peak lists or processed spectra: see annotation in “parameters” file. The mouse uniprot reference database “complete proteome” obtained on January 2nd, 2013 was used.

Project description:TiO2 enriched phosphopeptides of Iloprost stimulated platelets were digested with trypsin and labeled with iTRAQ. Samples were analysed by nano LC-MS/MS on an LTQ OrbitrapVelos (Thermo Scientific) coupled to an Ultimate 3000 Liquid Chromatography system (Dionex). Peptides were concentrated on a self-packed 100 µm ID reversed-phase (RP) trapping column (Kinetex C18, 2 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) in 0.1% TFA followed by separation on a self-packed 75 μm ID RP column (Kinetex C18, 30 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) using a binary gradient (solvent A: 0.1% FA and solvent B: 0.1% FA, 84% ACN) ranging from 3-45 % solvent B in 245 min at a flow rate of 230 nl/min. To minimize memory and carry-over effects from previous samples, an LC-wash program with high organic content was used prior to sample injection (Burkhart et al 2011). Survey scans were acquired in the Orbitrap with a resolution of 30,000 using the polysiloxane m/z 371.101236 as lock mass5 and MS/MS scans of the five most intense signals were acquired in the Orbitrap using HCD fragmentation (activation 0.1 ms, normalized collision energy of 50, isolation width of 1.5 m/z) with a resolution of 7,500. To compensate for elevated peptide charge states due to iTRAQ labels, a reaction tube containing 5 % ammonium water was placed in front of the ion source as described by Thingholm et. al. Database search and data processing: Raw data were processed with Proteome Discoverer 1.3 (Thermo Scientific) using Mascot (2.4) and Sequest for database search against the human Uniprot database (November, 4th 2010; 20,260 sequences) using the following search settings for both algorithms: i) trypsin with a maximum of two missed cleavages, (ii) carbamidomethylation of Cys (+57.0214 Da), peptide N-terminus iTRAQ, Lys iTRAQ (+144.102 Da) as fixed modifications and (iii) oxidation of Met (+15.9949 Da), Tyr iTRAQ (+144.102 Da) as well as phosphorylation of Ser/Thr/Tyr (+79.9663 Da) as variable modifications; (iv) MS and MS/MS tolerances of 10 ppm and 0.02 Da, respectively. A false discovery rate (FDR) of <1% was applied using the Peptide Validator node. Additionally, phosphorylation site localizations were validated by the phosphoRS node. Only phosphopeptide hits with <1% FDR of all validated peptides were considered for data interpretation and site assignment probabilities of >90 % (phosphoRS) were defined as confident. Data interpretation: To compensate for systematic variations or technical errors for each sample set, reporter areas were normalized based on modellized normal distribution with a +- 34% quantil. Therefore median iTRAQ ratios were calculated considering all unique <1% FDR peptides and normalization factors against the control sample were determined. For each peptide, all MS/MS spectra of one biological replicate were grouped in regard to modified site in protein and phosphoRS site localization (peptide groups) to determine the respective median ratios. Finally, for each peptide group, median values were calculated considering all three biological replicates. Peptides with ratios below 0.5 or above 2.0 were considered as regulated. Regulated phosphopeptides were searched against GPS 2.1 for kinase consensus site prediction. Values of high confidence (<2 % error) for PKA were taken into account.

Project description:Pilotexp_IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_eluate_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The enriched phosphopeptides were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID (MSA) and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. IMAC_TiO2_FT_ATM_ATR_dep_phos_15min_postIR: Proteins were isolated from 4 different conditions of Arabidopsis thaliana plants: wild type untreated, wild type irradiated, atm/atr mutant untreated, atm/atr mutant irradiated The proteins were tryptically digested and labelled each with one channel ofthe iTRAQ4plex reagent. The samples were mixed and subjected to phosphopeptide enrichment by IMAC followed by TiO2. The unphosphorylated peptides found in the flowthrough of the TiO2 column were separated by SCX and each fraction was analysed by nano-reversed phase HPLC coupled online to a LTQ Orbitrap Velos. Each peptide was fragmented both by CID and HCD, using the iontrap-MS analyser for CID and the Orbitrap detector for HCD scans. The iTRAQ ratio obtained for the unmodified peptides from this flowthrough sample was used to calculate protein ratios for the 4 different conditions.

Project description:In the current study, we used a mass spectrometry-based redox proteomics approach to test responses of the cysteine (Cys) proteome to selective disruption of the Trx- and GSH-dependent systems. Auranofin (ARF) was used to inhibit Trx reductase without detectable oxidation of the GSH/GSSG couple, and buthionine sulfoximine (BSO) was used to deplete GSH without detectable oxidation of Trx1. Results for 606 Cys-containing peptides (peptidyl Cys) showed that 36% were oxidized over 1.3-fold by ARF, while BSO-induced oxidation of peptidyl Cys was only 10%. Mean fold oxidation of these peptides was also higher by ARF than BSO treatment. Analysis of potential functional pathways showed that ARF oxidized peptides associated with glycolysis, cytoskeleton remodeling, translation and cell adhesion. Of 60 peptidyl Cys oxidized due to depletion of GSH, 41 were also oxidized by ARF and included proteins of translation and cell adhesion but not glycolysis or cytoskeletal remodeling. Studies to test functional correlates showed that pyruvate kinase activity and lactate levels were decreased with ARF but not BSO, confirming the effects on glycolysis-associated proteins are sensitive to oxidation by ARF. These data show that the Trx system regulates a broader range of proteins than GSH system, support distinct function of Trx and GSH in cellular redox control, and show for the first time in mammalian cells selective targeting peptidyl Cys and biological pathways due to deficient function of the Trx system. ICAT-labeled Cys peptides were analyzed by reverse-phase liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Peptide eluents were monitored in an MS survey scan followed by ten data-dependent MS/MS scans on an LTQ-Orbitrap ion trap mass spectrometer (Thermo Finnigan, San Jose, CA). The LTQ was used to acquire MS/MS spectra (2 m/z isolation width, 35% collision energy, 5,000 AGC target, 200 ms maximum ion time). The Orbitrap was used to collect MS scans (300-1600 m/z, 1,000,000 AGC target, 500 ms maximum ion time, resolution 30,000). All data were converted from .raw files to the .dta format using ExtractMS version 2.0 (Thermo Finnigan, San Jose, CA). The acquired MS/MS spectra were searched against a concatenated target-decoy human RefSeq (release 37 - September 2009 - 38108 target proteins) database of the National Center for Biotechnology Information using the SEQUEST Sorcerer algorithm (version 3.11, SAGE-N) searching parameters included partially tryptic restriction, parent ion mass tolerance (+/- 20 ppm), dynamic modifications of oxidized Met (+15.9949 Da), differential ICAT-modified Cys (+9.0302 Da) and static ICAT modification of Cys (+227.1270 Da). The peptides were classified by charge state and tryptic state (fully and partial) and first filtered by mass accuracy (10 ppm for high-resolution MS), and then dynamically by increasing XCorr and deltaCn values to reduce protein false discovery rate to less than 1%, according to the target-decoy strategy.

Project description:In order to identify molecular targets of USP2-45 in the small intestine, we carried out a quantitative proteomics iTraq (Isobaric tag for relative and absolute quantitation) screen to compare proteins from WT and knockout mice. With this analysis we identified, among others, the scaffolding protein Na(+)/H(+) Exchange Regulatory cofactor NHERF4 to be up-regulated (4.81-fold) in the duodenum of Usp2-KO mice. Methods : Usp2-KO and WT littermates males were entrained to 12h L:12h D for at least two weeks in light-light housing boxes. Mice were sacrificed by cervical disolcation at ZT13 under red dim light and duodenal mucosa was dissected by scraping. The tissue samples were quickly frozen in liquid nitrogen before total protein extraction. Proteins were extracted in a lysis buffer containing 8M Urea, 20mM Tris-HCl, pH 7.5, 14 µg/ml Aprotinin, 0.7 µg /ml PepstatinA 0.7 µg /ml Leupeptin, 1 mM NaVO4, 10 mM Na-Pyrophosphate, 50 µM MG132 and 1mM PMSF. Protein concentration was measured by Bradford quantitation. Relative quantitation of proteins in 4 pools of 3 KO and 4 pools of 3 WT tissue samples was carried out by iTRAQ (Isobaric Tags for Relative and Absolute Quantitation). After reduction and alkylation of cysteines, proteins were precipitated, redissolved in 8M Urea buffer and digested with trypsin (1:50 w:w) overnight. 80ug of digested material for each sample was labeled by reaction with one vial of iTRAQ 8-plex reagent (ABSciex). Samples were pooled , desalted and fractionated by off-gel electrofocusing. The 24 fractions obtained were analysed by nanoflow liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) on a hybrid linear trap LTQ-Orbitrap Velos mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) on a two-hour gradient. Full MS survey scans were performed at 60’000 resolution and the ten most intense multiply charged precursor ions detected in the full MS survey scan were selected for HCD (Higher energy Collision Dissociation) fragmentation and Orbitrap analysis (7500 resolution). Each spectrum was acquired at two relative collision energies (35% and 45%) and the spectra were summed. Data files were analysed with MaxQuant 1.3.0.5 incorporating the Andromeda search engine. Further data analysis was performed using the R statistical programming language version 2.15.2 (R core team 2012). Intensities for the reporter ions 113 to 121 were normalized using the Variance Stabilizing method (R package version 3.26.0). Proteins with non-zero intensities in all channels were evaluated for differential expression using the Local-Pooled-Error method (R package version 1.32.0) followed by correction for multiple testing according to Benjamini and Hochberg.

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