Project description:In this study, iTRAQ technique is employed to reveal the proteomic response of Providencia. sp under selenium and manganese stress. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for TMT kit/iTRAQ kit. Then, The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Agilent 300Extend C18 column. Finally, The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC.
Project description:Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS. Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins: WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?. Enzyme digestion, mass spectrometry and protein identification: The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria. Bioinformatics analysis: The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome. Keywords: other
Project description:For the analysis of chromosome 11-centric human proteome, we analyzed the human brain hippocampal proteome. The samples from brain tissues were extensively off-gel fractionated by 12 fractions and analyzed by a high resolution mass spectrometer, Orbitrap XL ETD with collision induced dissociation, after LC separation using C18 column.
Project description:We analyzed methylome changes in Arabidopsis between seed development (2 stages corresponding to Bent (B) and mature (MS) embryo stages) at three growing conditions (23, 25 and 27°C)
Project description:We analyzed transcriptome changes in Arabidopsis between seed development (3 stages corresponding Heart (H), Bent (B) and mature (MS) embryo stages) at three growing conditions (23, 25 and 27°C)
Project description:Four different developmental time points representing the Afu development from dormant conidia into young hyphae (0h, 4h, 6h, 8h) were chosen. Cell walls of the Afu samples were extracted using denaturing methods followed by isolation of solubilized proteins. Samples were processed using filter aided sample preparation (FASP) / trypsin digestion. The peptides were analyzed by one-dimensional LC (capillary C18 nano-Eflow mode) on-line with a nano-ESI source and tandem MS (LTQ ion trap).
Project description:We performed 4D label free-based quantitative proteomic analysis of Eimeria tenella unsporulated oocysts and sporulated oocysts. Briefly, protein extraction was performed using liquid nitrogen grinding and sonication on ice in SDT buffer (4% SDS, 100 mM Tris-HCl, pH7.6). After trypsin digestion, the peptides were separated on Easy-nLC1000 liquid chromatograph (Thermo Fisher Scientific, Waltham, MA, USA) with a trap column (Thermo Fisher Scientific Acclaim PepMap100, 100 μm × 2 cm, nanoViper C18) and analytical column (Thermo Fisher Scientific Easy Column, 25 cm long, 75 μm inner diameter, 1.9 μm resin). LC-MS/MS analysis was conducted on a timsTOF Pro mass spectrometry. The resulting LC-MS/MS data files were processed with the MaxQuant software (version 1.5.3.17) for identification and quantitation analysis.
Project description:To identify low abundance autocrine growth factors in CHO cell conditioned media, we utilized a label-free shotgun proteomics approach. CHO cell conditioned media were harvested from fed batch bioreactors and concentrated using methanol/chlorofrom precipitation. Proteins in the samples were then subjected to proteolysis with trypsin, and then subjected to primary fractionation using a SCX column, followed by RP liquid chromatography MS (LC-MS) with a LTQ Orbitrap Velos instrument using the data dependent acquisition (DDA) method. The MS system was set up and run with a method which enabled fast acquisitions of high quality peptide precursor and fragment ion data, with the desired precursor mass accuracy of ±5 ppm. For the LTQ Orbitrap Velos MS, the data-dependent MS/MS analytical workflow in positive ion mode was used. Each precursor survey scan (m/z: 300 to 1800) by the Orbitrap mass analyzer (resolution = 60,000 FWHM) was linked to 10 MS/MS events using the 2D ion trap CID approach, with dynamic ion exclusion set at 60 s. This value was determined based on the observed mean peptide chromatographic peak width. All other instrument parameters were set up according to the manufacturer’s suggested values for complex peptide samples. The nano-ESI source was fitted with a 30-µm stainless steel nano-bore emitter (Thermo Fisher Scientific) with 1.7 kV applied near the tip. Raw data files from the LTQ Orbitrap Velos MS were processed using the Proteome Discoverer 1.3 software (Thermo Fisher Scientific). The LC-MS data were searched against the human (Homo sapiens; UniProtKb, updated in August 2012, 45 848 entries), mouse (Mus musculus; UniProtKb, updated in August 2012, 31 528 entries) and chinese hamster (Cricetulus griceus; UniprotKb, updated in August 2012, 24 609 entries) protein databases using the Sequest search engine for the LTQ Orbitrap Velos LC-MS data, assuming tryptic digestion with precursor ions to fall within 10 ppm of projected m/z values and a fragment ion mass tolerance of 0.5 m/z. The specified search parameters were carbamidomethylation of cysteine as fixed modification, oxidation of methionine as dynamic modification and a maximum of two missed cleavage events. Reverse database searching resulted in a specific false discovery rate (FDR) of 1% at the peptide and protein level.
Project description:Twenty microliter aliquots of BALF samples were dissolved in SDS-sample buffer and applied onto a 4-12% Nupage gel with MOPs running buffer. The run stopped after the samples migrated approximately ¼ distance into the gel. Each lane of the gel was sliced into smaller pieces, and subjected to destaining, reducing/alkylation, and in-gel trypsin digestion. The extracted peptides were applied for LC-MS/MS analysis using either a Thermo Orbitrap Fusion or a Thermo Orbitrap Fusion Lumos operated with an in-line Thermo nLC 1200 and an EASY-Spray ion source. Pepetides were separated using a 2 cm Pepmap 100 C18 trap column and a 25 cm Easy-spray Pepmap 100 C18 analytical column. MS/MS data acquisitions were operated at a 120,000 resolution (m/z 200) with a scan range of 350-1950 m/z and CID fragmentation.