Protein profiles of extracellular vesicles from healthy preschool children born late preterm (LP) and full-term (T).
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q-Exactive
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Pierluigi Mauri Dario Di Silvestre
PROVIDER: MSV000086867 | MassIVE | Mon Feb 15 06:09:00 GMT 2021
REPOSITORIES: MassIVE
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2022-12-16 | MSV000090915 | MassIVE
Project description:Murine TEC for proteomic analyses were obtained from 3 pools of WT or AB0/0 mice, 3 mice per pool (5-6 week-old). TEC were isolated, enriched and processed through CD45+ cell-depletion as described above. CD45- cell-samples were pelleted and stored at -80 C until use.
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Project description:Human lung microvascular endothelial cells (HL-mECs) purchased from Lonza Clonetics (Walkersville, MD, USA) and cultured in EGM-2MV (Lonza) containing 10% (FBS). The proteomic analysis was performed on Mock-, SARS-CoV-2-, and UV-SARS-CoV-2-infected HL-mECs, collected at day 1, 2 and 3 p.i.
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2020-12-30 | MSV000086649 | MassIVE
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2022-02-24 | PXD012490 | Pride
Project description:This data displays both known and unknown extra-cellular proteins from 13 species of Lactic Acid bacteria found in the honey-crop of the honeybee Apis. mellifera mellifera. The tryptic peptides from the secreted proteins were run on an Agilent HPLC on a C18 reverse phase column (75 µm x 150 mm, particle size 3 µm). Total run time was 90 min and flow rate 300 nl/min. Buffers used for gradient was 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). The buffer mixing was 5 min 5% buffer B, followed by 5%-45% buffer B in a linear gradient for 50 min, followed by 45%-80% buffer B in a linear gradient for 5 min. The 80% of buffer B was then kept for 15 min and then rapidly back to 5% buffer B for the final 15 min. The fractions from HPLC were loaded on an LCQ Deca XP Plus Ion trap mass spectrometer (ThermoScientific). Genomic DNA were prepared from all 13 LAB strains depicted earlier and sequenced at MWG Eurofins Operon (Ebensburg, Germany) using Roche GS FLX Titanium technology from Roche (Basel, Switzerland). For each genome a shotgun library was constructed with up to 700,000 reads per segment and was generated by sequencing in 2x half segment of a full FLX+ run. Each genome had an 8 kpb long-paired end library constructed. Approximately 300,000 true paired end reads, sequence tags, and scaffolds with GS FLX+ chemistry using 2x half segment of a full run were generated. Clonal amplification was performed by emPCR in both library types. The sequencing was continued until 15-20 fold coverage was reached. The obtained reads were assembled by the software Newbler 2.6 from Roche (Basel, Switzerland). ORF prediction and automated annotation was performed at Integrated Genomics Assets Inc. (Mount Prospect, Illinois, USA). In ORF prediction three different software were used, GLIMMER, Critica, and Prokpeg. Automated annotation was performed with the ERGOTM algorithms (Integrated Genomics Assets Inc. Mount Prospect, Illinois, USA). The resulting mass spectra-files obtained from the mass spectrometry analysis were searched using MASCOT against a local database containing the predicted proteome of the 13 LAB. We used a cut off Ions score of 38 as a value for determining that the protein was identified. Individual ion scores that were greater than 38 indicated identity or extensive homology (p<0.05) of the protein. Protein sequence similarity searches were performed with software BLASTP in the software package BLAST 2.27+ against a non-redundant protein database at NCBI. Pfam (default database), and InterProScan (default databases). Expressed proteins identified by peptide mass fingerprinting were manually re-annotated.
2013-10-01 | PXD000187 | Pride
Project description:Washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 counts) with charge states >+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da Oxidation (M) and Carbamidomethyl (C) specified as variable modifications Enzyme specificity was trypsin, 1 missed cleavage was possible
2014-04-28 | PXD000505 | Pride
Project description:Bacteria were washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 counts) with charge states >+2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/MS were dynamically excluded for 30 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da; Oxidation (M) and Carbamidomethyl (C) specified as variable modifications, Enzyme specificity was trypsin, 1 missed cleavage was possible
2014-04-28 | PXD000504 | Pride
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%.
2013-06-20 | PXD000284 | Pride
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2016-08-08 | PXD001620 | Pride
Project description:Pretreated human follicular fluid was prepared for targeted metabolomics experiments. For this purpose, 12-point standard calibration curves in the respective range with reference material were measured, and the limit of detection (LOD) and limit of quantification (LOQ) for each neurotransmitter under investigation were calculated. LC-MS/MS was conducted by an ultra-performance liquid chromatography method using a Waters ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 µm, Waters, USA). The Waters acquisition UPLC was coupled to an API 4000 triple quadrupole mass spectrometer (AB SCIEX, USA). The mobile phase A buffer was 10% methanol in water (containing 0.1% formic acid), and the mobile phase B buffer was 50% methanol in water (containing 0.1% formic acid). The LC gradient elution was at a flow rate of 0.3 mL/min at 0-8.0 min and 0.4 ml/min at 8.0-17.5 min, then 10%-30% B at 0-6.5 min; 30%-100% B at 6.5-7 min; 100% B at 7-14min; and 100%-10% B at 14-17.5min. The injection volume was 5 µl and the column temperature was set to 40 °C. Twenty-three metabolites (Ach: Acetylcholine chloride; Gln: Glutamine; Glu: Glutamate; His: L-Histidine; NE: norepinephrine; Tyr:Tyrosine; Trp:Tryptophan; Kyn: Kynurenine; GABA: 4-Aminobutyric acid;HisA:Histamine; PA:Picolinic acid;TyrA:Tyramine;DA:Hydroxytyramine hydrochloride; TrpA:Tryptamine;5-HT:Serotonin hydrochloride;E:Adrenaline hydrochloride;KynA:Kynurenic acid;5-HIAA:5-Hydroxyindole-3-acetic acid;DOPA:Levodopa;XA:Xanthurenic acid;VWA:Vanillymandelic Acid; 5-HTTP:5-Hydroxytryptophan;MT:Melatonine)were simultaneously reported in LC-MS/MS multiple reaction monitoring (MRM) mode. Data analysis was performed with Analyst 1.6 software.
2022-08-12 | PXD031560 | Pride