Dataset Information

Iloprost (2/5 nM) stimulated Platelets

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Florian Beck

PROVIDER: PXD000242 | Pride | 2013-12-02

REPOSITORIES: Pride

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Time-resolved characterization of cAMP/PKA-dependent signaling reveals that platelet inhibition is a concerted process involving multiple signaling pathways.

Beck Florian F Geiger Jörg J Gambaryan Stepan S Veit Johannes J Vaudel Marc M Nollau Peter P Kohlbacher Oliver O Martens Lennart L Walter Ulrich U Sickmann Albert A Zahedi René P RP

Blood 20131209 5

One of the most important physiological platelet inhibitors is endothelium-derived prostacyclin which stimulates the platelet cyclic adenosine monophosphate/protein kinase A (cAMP/PKA)-signaling cascade and inhibits virtually all platelet-activating key mechanisms. Using quantitative mass spectrometry, we analyzed time-resolved phosphorylation patterns in human platelets after treatment with iloprost, a stable prostacyclin analog, for 0, 10, 30, and 60 seconds to characterize key mediators of pl ...[more]

PMID: 24324209

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Project description:Four bile samples collected from patients with a malignant (PAC, CC) or a nonmalignant (CP, BS) biliary stenosis were used for comparative proteomic analysis. Briefly, bile samples were centrifuged at 16,000/g/ for 10 min at 4 C. Each supernatant was delipided with Cleanascite (Biotech Support Group, North Brunswick, NJ, USA) and ultrafiltrated using a 3 kDa filter cut-off (YM-3 centricon, Millipore, Bedford, MA, USA). For each sample, 50 ug of proteins were mixed with 0.5 M triethylammonium bicarbonate (TEAB) buffer pH 8.0 to a total volume of 100 uL. One ug of bovine lactoglobulin (LACB) was spiked in each sample as an internal standard. Proteins were reduced and alkylated before digestion of N-linked oligosaccharides from glycoproteins using PNGase F (Sigma-Aldrich, St. Louis, MO, USA). Proteins were then digested with trypsin and the resulting peptides were tagged with one of the isobaric tags from the iTRAQ reagents 4plex kit (AB Sciex, Foster City, CA, USA) according to the manufacturer's instructions. The four samples were then pooled and dried under vacuum. Labeled peptides were fractionated according to their isoelectric point on an Agilent 3100 OFFGEL fractionator using 24 cm pH 3-10 linear immobilized pH gradients (GE Healthcare, Chalfont St. Giles, UK) following manufacturer's instructions. Fractions were then recovered in separate tubes for high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). MS analysis was performed on a LTQ Orbitrap XL from Thermo Electron (San Jose, CA, USA) equipped with a NanoAcquity HPLC system from Waters. Peptides were trapped on a home-made 5 um 200 A Magic C18 AQ (Michrom, Auburn, CA, USA) 0.1 x 20 mm pre-column and separated on a home-made 5 um 100 A Magic C18 AQ (Michrom) 0.75 x 150 mm column with a gravity-pulled emitter. The analytical separation was run for 65 min using a gradient of H_2 O/formic acid (FA) 99.9%/0.1% (solvent A) and CH_3 CN/FA 99.9%/0.1% (solvent B). The gradient was run at a flow rate of 220 nL/min as follows: 5% B for 1 min, from 5 to 35% B in 54 min, from 35 to 80% B in 10 min. For MS survey scans, the OT resolution was set to 60,000 and the ion population was set to 5 x 10^5 with an m/z window from 400 to 2000. Maximum of 3 precursors were selected for both collision-induced dissociation (CID) in the LTQ and high-energy C-trap dissociation (HCD) with analysis in the Orbitrap (OT). For MS/MS in the LTQ, the ion population was set to 1 x 10^4 (isolation width of 2 m/z) while for MS/MS detection in the OT, it was set to 2 x 10^5 , with resolution of 7,500, first mass at m/z = 100, and maximum injection time of 750 ms. The normalized collision energies were set to 35% for CID and 60% for HCD. Peak lists from MS analysis were searched against Uniprot_Swissprot database (version 2011_2) using Phenyx protein identification software (GeneBio, Geneva, Switzerland). /Homo Sapiens/ taxonomy was specified for database searching. The parent ion tolerance was set to 10 ppm. Variable amino acid modifications were oxidized methionine and deamidated asparagine. Carbamidomethylation of cysteines and iTRAQ-labeled peptides on the amino terminus and lysine were set as fixed modifications. Trypsin was selected as the enzyme, with one potential missed cleavage, and the normal cleavage mode was used. Only one search round was used with selection of "turbo" scoring. The peptide p value was 10^-2 for LTQ-OT data. Protein and peptide scores were set up to maintain the false positive rate below 5%.

Project description:Application of a novel TiO2-based phosphopeptide enrichment protocol to study the role of protein arginine phosphorylation in B.subtilis. 1 iTRAQ Quantification of pArg peptides in B.subtilis upon heat shock and oxidative stress in comparison to unstressed control 2 Studying pArg upon deletion of McsB protein arginine kinase in B.subtilis (double mutant cells for McsB and YwlE phosphoarginine phosphatase) 3 Studying pArg in wt cells upon inhibition of YwlE phosphatase with cell wall permeable inihbitor sodium pervanadate Short overview McsB is a bacterial protein arginine kinase that is involve in stress adaptation for heat shock and oxidative stress - cells were grown in LB medium until O.D. 0.6 and split into 3 fractions - one fraction was treated with heat stress for 20 min at 50 degrees C, one was stressed with 1mM diamide (induced oxidative foldeing stress), and one was kept at 37 degrees C (control) - cell harvest, lysis with a modified FASP protocol including tryptic cleavage - iTRAQ labelling for some experiments - TiO2 enrichment to purify all phosphopeptides - LC-MS/MS analysis of phosphopetides - phosphosite localization by phosphoRS and manually - quantitation of phosphorylation relative to protein abundance in the different samples. Peptide and protein identification: Raw data were extracted by the Protein Discoverer software suite (version 1.2.227 for wt phosphorylation, all 4 replicates of the iTRAQ/TiO2 pArg study and replicates 1 and 2 for the pervanadate and the double mutant experiments and version 1.3.0.339 for protein abundance determination, and replicates 3 and 4 of the pervanadate and double mutant experiments. Thermo-Fisher Scientific) and searched against a combined forward/reversed database of B. subtilis protein sequences and common contaminants using MASCOT (version 2.2, www.matrixscience.com). Since cells were grown in LB medium, all raw data were independently searched against a combined B. subtilis and yeast (S. cerevisae) database which did not result in a significant increase in protein hits or identification of yeast proteins. Carbamidomethylation of cysteine, iTRAQ modification of peptide Nterminus and lysine;-amino group were set as fixed modifications. 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Project description:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.

Project description:Abstract still has to be written. The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Samples were first loaded on a trapping column (made in-house, 100 um internal diameter (I.D.) x 20 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 um I.D. x 150 mm, 5 um beads C18 Reprosil-HD, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and were separated with a linear gradient from 2% solvent A' (0.05% formic acid) to 55% solvent B' (0.05% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min followed by a wash reaching 100% solvent B'. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the six most abundant peaks in a given MS spectrum. Full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The six most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 60 s. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts. From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.3.01, Matrix Science). While generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 s and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal to noise limit was set at 2. These peak lists were then searched with the Mascot search engine (Matrix Science) using the Mascot Daemon interface (version 2.3, Matrix Science). Spectra were searched against the human (H. sapiens) Swiss-Prot database. 13C2D3-acetylation of lysine side-chains, carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide were set as fixed modifications for the N-terminal COFRADIC analyses. Variable modifications were 13C2D3-acetylation and acetylation of protein N-termini. Pyroglutamate formation of N-terminal glutamine was additionally set as a variable modification. Mass tolerance on precursor ions was set to 10 ppm (with Mascot's C13 option set to 1) and on fragment ions to 0.5 Da. Endoproteinase semi-Arg-C/P (Arg-C specificity with arginine-proline cleavage allowed) was set as enzyme allowing no missed cleavages. The peptide charge was set to 1+, 2+, 3+ and instrument setting was put to ESI-TRAP. Only peptides that were ranked one and scored above the threshold score, set at 99% confidence, were withheld. Quantification of the degree of Nt-Acetylation was performed as described previously (1). All data management was done in ms_lims (2).

			Action	DRS
	2nMIloprostRep1.pep.xml	Pepxml
	2nMIloprostRep1L1.msf	Msf
	2nMIloprostRep1L1.mzML	Mzml
	2nMIloprostRep1L23.msf	Msf
	2nMIloprostRep1L23.mzML	Mzml