Identification of peptides and proteins in insect based feed ingredients using LCMSMS
Ontology highlight
ABSTRACT:
INSTRUMENT(S): maXis
ORGANISM(S): Alphitobius Diaperinus (ncbitaxon:27448) Hermetia Illucens (ncbitaxon:343691) Acheta Domesticus (ncbitaxon:6997) Tenebrio Molitor (ncbitaxon:7067)
SUBMITTER: 
Josef Daniel Rasinger
PROVIDER: MSV000083737 | MassIVE | Wed May 01 20:05:00 BST 2019
REPOSITORIES: MassIVE
Similar Datasets
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.
2022-12-18 | PXD038884 | iProX
Project description:Carfilzomib-sensitive (AMO1) and resistant (AMO1-CFZ) MM cells were grown in RPMI-1640 medium supplemented with 10 % FBS and 0.68 mM L-glutamine, in a humidified incubator at 5% CO2 and 37 °C. The AMO1-CFZ medium was additionally added 90 nM CFZ, while the CFZ sensitive AMO1 cells were added vehicle (0.009 % DMSO) only. AMO1-CFZ was either harvested directly or grown for 1 week in CFZ free medium (vehicle only) prior to harvest. AMO1 and carfilzomib-resistant AMO1-CFZ cells were resuspended in 100 µl 1% sodium deoxycholate, 100 mM Tris-HCl pH 8.5, 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM chloroacetamide (CAA), heated at 90 °C for 45 min and sonicated for 10 cycles (30 s ON/30 s OFF) using a Bioruptor pico sonicator. After centrifugation at 16000 × g for 10 min, 50 µg soluble protein from each sample was added 100 µl 0.1M ammonium bicarbonate, 0.5 µg trypsin and digested overnight at 37°C. Peptides were desalted using C18 spin columns, dried in a speedvac centrifuge and resuspended in 0.1% formic acid prior to MS analysis. Label-free quantitatative (LFQ) LC-MS/MS was performed on a timsTOF Pro (Bruker Daltonics) connected to a nanoElute (Bruker Daltonics) HPLC. Peptides were separated over a Bruker PepSep C18 (75 µm × 15 cm) column with running buffers A (0.1 % formic acid) and B (0.1 % formic acid in acetonitrile) using a 100 min gradient from 2 % B to 40 % B. The timsTof instrument was operated in the DDA PASEF mode with 10 PASEF scans per acquisition cycle and accumulation and ramp times of 100 ms each. The ‘target value’ was set to 20000 and dynamic exclusion was activated and set to 0.4 min. The quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th for m/z > 800.
2024-06-07 | PXD045439 | Pride
Project description:Cultured mouse lymphoma cells were lysed with a buffer containing 50 mM Tris-HCl (pH 8.0) and 8 M Urea with or without a protease inhibitor cocktail (Sigma). The lysates were desalted and digested with sequencing-grade modified trypsin (Promega). Different amounts of peptides were analyzed using nanoflow RPLC-MS/MS on a linear ion trap mass spectrometer (LTQ XL, Thermo Electron, San Jose, CA). The raw MS/MS data were searched using SEQUEST running under BioWorks (Rev. 3.3.1 SP1) (Thermo Electron, San Jose, CA) against a mouse IPI proteome database (Version 3.62) downloaded from the European Bioinformatics Institute (EBI) (http://www.ebi.ac.uk). For the SEQUEST analysis, the peptide mass tolerance was set as 2.0 Da and the fragment ion tolerance was 1.0 Da. A tryptic enzyme restriction with a maximum of two internal missed cleavage sites was used. The Xcorr versus charge state values were filtered at Xcorr ≥ 1.7 for [M+H]1+ ions, ≥ 2.5 for [M+2H]2+ ions, ≥ 3.2 for [M+3H]3+ ions, and P ≤ 0.01 and ΔCn ≥ 0.08 were applied for identification of all fully tryptic peptides. The peptide ion chromatogram was extracted using a minimum intensity threshold of 100, mass tolerance of 2.0 amu and smoothing point of 5, and the area of the extracted ion chromatographic (XIC) peak was integrated and calculated using the PepQuan module in Bioworks (Thermo Electron, San Jose, CA).
2022-02-15 | PXD000334 | Pride
Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.
2022-12-07 | PXD023141 | Pride
Project description:A549 cells were transfected with 100 pmol DNA probes complementary to the back-splice site of circITGB6 or PDPN 3’UTR region. 24 h later, cells were washed with PBS and lysed with the lysis buffer (150 mM NaCl, 20 mM Tris-HCl, pH 7.5, 1.5 mM MgCl2, 0.5% NP-40, 0.5% Triton X-100, 10% glycerol, RNase inhibitors (Thermo Scientific), protease and phosphatase inhibitor cocktails). Pre-treated streptavidin magnetic beads were incubated with cell lysates at 4°C for 1 h, followed by washing five times with the washing buffer (20 mM Tri-HCl, pH7.5, 1 mM EDTA and 450 mM NaCl) and elution with Laemmli sample buffer. The circITGB6- or PDPN mRNA- interacting proteins were subjected to SDS-PAGE and visualized by coomassie blue staining or immunoblot analysis. Protein bands were excised and identified by LC-MS/MS mass spectrometry and Proteome Discoverer software (version 1.4; Thermo Scientific).
2023-10-13 | PXD043341 | Pride
Project description:The present study was initiated to evaluate the quantitative proteomic profiling of Protothecazopfiigenotypes. The cells (P.zopfii genotype 1 –noninfectious and genotype 2 -infectious) were cultured in triplicates until the mid-logarithmic growth phase; the proteins were extracted after sonication on ice, followed by 2D DIGE separation as recommended by the manufacturer (GE Healthcare). The differentially expressed proteins spots were identified using Decodon software analysis (Delta 2D version 4.0 software). Protein identification was carried out by MALDI TOF MS/MS (Ultraflex III TOF/TOF, Bruker Daltonics, Bremen, Germany). The spectra was acquired using the automated option (AutoXecute ) of the Flex Analysis software version 3.3 (Bruker Daltonics, Leipzig, Germany), processed using Flex analysis software version 3.3 (Bruker Daltonics, Leipzig, Germany) and the database search was conducted using the MS/MS ion search (MASCOT, http://www.matrixscience.com) against all entries of NCBInr (GenBank)/Swissprot with subsequent parameters: trypsin digestion, up to one missed cleavage, fixed modifications: carbamidomethyl and with the following variable modifications: oxidation (M), peptide tol.: +- 1.2 Da, MS/MS tol.: +- 0.6 Da, peptide charge: +1. The results were assessed using MOWSE score, p- and E values and those possess positive hits with cRAP database were eliminated from the list.
2013-07-22 | PXD000126 | Pride
Project description:Tissue lysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012)) Briefly, greater than 300 mg of frozen tumor mass was homogenized and sonicated in urea lysis buffer (20 mM HEPES pH 8.0, 9 M urea, 2.5 mM sodium pyrophosphate, 1.0 mM beta-glycerophosphate, 1% N-octyl glycoside, 2 mM sodium orthovanadate). Total protein was measured using the BCA Protein Assay Kit (Thermo Scientific/Pierce) and 25 mg of total protein was used for phospho-proteomic analysis. Phospho-tyrosine peptide enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012); Rubbi, L., et al. Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling. Sci Signal 4, ra18 (2011); Graham, N.A., et al. Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death. Molecular systems biology 8, 589 (2012)) Phospho-peptides were identified using the Proteome Discoverer software (version 1.3.0.339, Thermo Fisher Scientific). MS/MS fragmentation spectra were searched using SEQUEST against the Uniprot human reference proteome database with canonical and isoform sequences (downloaded January 2012 from uniprot.org). Search parameters included carbamidomethyl cysteine (*C) as a static modification. Dynamic modifications included phosphorylated tyrosine, serine, or threonine (pY, pS, pT, respectively) and oxidized methionine (*M). The Percolator node of Protein Discoverer was used to calculate false-discovery rate (FDR) thresholds and the FDR for the datasets was adjusted to 1% (version 1.17, Thermo Scientific). The Percolator algorithm uses a target-decoy database search strategy and discriminates true and false identifications with a support vector machine. The PhosphoRS 2.0 node was used to more accurately localize the phosphate on the peptide44. Only phospho-peptides with at least one phospho-tyrosine assignment with a reported probability above 20% were considered. MS2 spectra for all reported phosphopeptides are available under the PRIDE accession numbers
2013-11-21 | PXD000238 | Pride
Project description:Cyanobacteria extracts and SPE fractions. LC MSMS analysis was performed in an UltiMate 3000 UPLC system (Thermo Scientific) using a Kinetex 1.7 mm C18 reversed phase UHPLC column (50 X 2.1 mm) and Maxis Impact HD Q-TOF mass spectrometer (Bruker Daltonics) equipped with ESI source.
2019-10-10 | MSV000084447 | MassIVE
Project description:Phosphoproteome analysis of HL-1 cardiomyocytes carrying AKT1 or AKT2 isoform specific knock down, respectively. Six biological replicates were analysed. Cells were grown until reaching 90 % confluency, starved for 50 minutes followed by stimulation with 200 nM insulin for 10 minutes. In solution stable isotope dimethyl labeling was performed following the protocol of Boersema et al. (2009) Nat. Protoc. 4, 484-494 Fe-NTA Phosphopeptide Enrichment Kit (Thermo Scientific) was used according to the manufacturer’s instructions. Peptides were desalted with Graphite Spin Columns (Thermo Scientific) and fractionated by automated off-line 2-D LC. First dimension: 1 mm × 15 cm Polysulfoethyl-Aspartamide column (Dionex, Thermo Scientific). Two replicates were alternatively fractionated in-solution using the Agilent 3100 OFFGEL Fractionator (24 cm IPG strips pH 3-10 (GE Healthcare) and a 24 well frame set from Agilent Technologies). Peptides from the single SCX fractions were further separated and analyzed by reversed-phase nano-LC-MS/MS. Sequence database-search of the MS data was performed against the uniprot mouse database (downloaded 13.06.2012) using MaxQuant (Version 1.3.0.5).
2014-08-28 | PXD000268 | Pride
Project description:To determine what signaling pathways are affected by I3MO in MM cells, 5 MM cell lines including ANBL6, ANBL6 BR, ARP1, RPMI-8226, and U266, were cultured with DMSO or 5/10µM I3MO for 48 h. Total RNAs of 2 x 10^6 cells for each sample were extracted using the RNeasy Mini Kit (Qiagen). The RNA yield was determined by Nanodrop technology (Thermo Fisher Scientific), and quality was verified on the Agilent 2100 bioanalyzer (Agilent Technologies). cDNA libraries were prepared for sequencing using standard Illumina protocols, followed by sequencing on the NextSeq500 Sequencing System (Illumina). We used gene expression profiling data to determine differential expression of genes in MM cells in culture with DMSO or I3MO.
2022-01-26 | GSE194379 | GEO