Dataset Information

Shewanella oneidensis outer membrane and outer membrane vesicles

ABSTRACT: This project compares the outer membrane proteome to the proteome of outer membrane vesicles of the bacterium Shewanella onedensis. S. oneidensis outer membrane was purified via the sarkosyl method described in (Brown, 2010). A 50 mL overnight culture of cells were harvested by centrifugation at 10,000× g for 10 min. cell pellet suspended in 20 mL of 20 mM ice-cold sodium phosphate (pH 7.5) and passed four times through a French Press (12000 lb/in2). The lysate was centrifuged at 5,000× g for 30 min to remove unbroken cells. The remaining supernatant was centrifuged at 45,000 × g for 1 h to pellet membranes. Crude membranes were suspended in 20 mL 0.5% Sarkosyl in 20 mM sodium phosphate and shaken horizontally at 200 rpm for 30 min at room temperature. The crude membrane sample was centrifuged at 45,000 × g for 1 h to pellet the OM. The pellet of OM was washed in ice-cold sodium phosphate and recentrifuged. The cells were pelleted by centrifugation at 5000 x g for 20 min at 4°C, and the supernatant was filtered through 0.45 μm pore size filters to remove remaining bacterial cells. Vesicles were obtained by centrifugation at 38,400 x g for 1 h at 4°C in an Avanti J-20XP centrifuge (Beckman Coulter, Inc). Pelleted vesicles were resuspended in 25 ml of 50 mM HEPES (pH 6.8) and filtered through 0.45 μm pore size filters. Vesicles were again pelleted as described above and finally resuspended in 50 mM HEPES, pH 6.8. Extracellular DNA, flagella, and pili can all be co-purified. Protocol was adapted from (Perez-Cruz, 2013).

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Shewanella Oneidensis (strain Mr-1)

SUBMITTER: Lori Zacharoff

LAB HEAD: Mohamed El-Naggar

PROVIDER: PXD020577 | Pride | 2021-11-10

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

			Action	DRS
	lori_membraneA_15May181.raw	Raw
	lori_membraneB_15May181.raw	Raw
	lori_membrane_8March18.raw	Raw
	lori_vesicleA_15May18.raw	Raw
	lori_vesicleB_15May18.raw	Raw

Items per page:

1 - 5 of 7

Similar Datasets

Project description:Prefrontal cortex tissue slices from a cognitively and neurpathologically normal human subject were obtained from the University of Pennsylvania (UPenn) Brain Bank. 300-350 mg grey matter was homogenized in 1.5 ml solution A (0.32 M sucrose, 1mM MgCl2 and 0.1mM CaCl2) with a Teflon pestle. ~100 ?l of the homogenate was saved, solubilized with 1% SDS and clarified by centrifugation. To prepare the [13C6]brain ISTD, 400 mg labeled MouseExpress brain tissue (Cambridge Isotopes, MA) was homogenized in 4 ml buffer A as described above, centrifuged at 1,000 x g for 10 min to clarify, agitated on a rocker at 4�C for 30 min, and centrifuged at 10,000 x g for 20 min. The pellet, a crude membrane fraction, was dissolved in 2 ml 20 mM Tris pH 7.4 with 1% SDS. Total protein in all preparations was quantified with the micro BCA assay (Pierce) and all solutions were supplemented with protease and phosphate inhibitor cocktails (Sigma) as well as 1 mM sodium fluoride and sodium orthovanadate (Sigma). Human cortex homogenate was mixed with the [13C6]brain ISTD (1?g/?l) at a ratio of 2:1(?g/?g) and processed for LC-MS/MS: 40 ?l preparations were heated with lithium dodecyl sulfate (LDS) (Invitrogen) buffer at 95�C for 20 min, separated on a 1.5 mm 4-12% Bis-Tris Gel (Invitrogen), cut into five fractions, chopped into ?2 mm cubes, washed in 200 ?l 50% acetonitrile (ACN) containing 25 mM NH4HCO3, reduced in 300 ?l 10 mM DTT, alkylated in 300 ?l 55 mM iodoacetamide (Sigma), and digested with 80-120 ?l trypsin (.025 ?g/?l) (Promega) overnight at 37�C, so that the amount of trypsin was 1:5 of the total protein to be digested by mass. Peptides were recovered from gel cubes into 200 ?l 50/50 H2O/ACN with 3% formic acid by vortex-mixing and sonication for 20 min each, twice. Samples were then evaporated to a 100 ?l volume, brought to 1ml in H20 with 0.1% formic acid, desalted with Oasis� HLB cartridges (Waters), evaporated almost to completion and suspended in ~ 45?l H2O with 0.1% formic acid, and filtered with 0.22 �m Ultra free-MC filter cartridges (Millipore). LC-data dependant -MS/MS analyses were conducted on a Q Exactive (ThermoFisher Scientific) quadrupole orbi-trap hybrid instrument with an Easy nLC-II (ThermoFisher Scientific) nano-pump/autosampler. 3?l peptide sample (~ 1 ug) was loaded and resolved on a 20 cm x 75 �m proteopepII C18 packed tip column over a 90 min gradient at a flow rate of 350 nL/min, 0-35% mobile phase B (ACN, 0.1 formic acid). A data-dependent top 10 method was used to acquire a 70,000 resolution full scan to trigger ten 17,500 resolution HCD scans. Ions were isolated for MS/MS analysis with a 2.0 Da window on the quadrupole. Average cycle times were 1.2 sec. Each sample was analyzed in triplicate. Raw files from triplicate injections were searched together within Proteome Discoverer 1.3 (ThermoFisher Scientific) using SEQUEST and the human refseq. database (release 47, ftp://ftp.ncbi.nih.gov/refseq/H_sapiens/mRNA_Prot/). Search parameters allowed trypsin to cleave after Lysine and Arginine and have 2 missed cleavages. Precursor ion mass tolerance was set to 15 ppm and fragment ion mass tolerance was 20 mmu. The dynamic modification of methionine (oxidation = 15.995 Da) and lysine (13C6 = 6.020 Da), in addition to the static modification of cysteine (carbamidomethylation = 57.021 Da) was accepted on up to 4 residues per peptide. Within the Proteome Discoverer Software, SILAC pairs were identified using the �2 plex� workflow node, and all peptides were rescored using the Percolator algorithm node. Finally, peptides were filtered at 1% false discovery rate (FDR).

Project description:High activation of the PI3K-AKT-mTOR pathway is characteristic for T-cell acute lymphoblastic leukemia (T-ALL). Here we investigated how 4EGI-1, a small inhibitor of cap-dependent translation, induces apoptosis in T-ALL clones displaying hyperactive PI3K-AKT-mTOR signaling. 4EGI-1 treatment reduced the ribosomal occupancy of transcripts that define the molecular difference between T-ALL blasts and normal thymocytes. These transcripts encoded proteins for the translational machinery, for mitochondrial metabolism as well as oncoproteins such as Cyclin D1, Bcl-2 and c-myc. Therefore, targeting translation initiation proves valuable in situations where mTOR is activated by multiple events. For each biological replicate 5 x 107 #483 T-ALL cells were treated with 100 ÂµM 4EGI-1 or DMSO. After 2 h incubation at 37Â°C cells were washed in ice-cold PBS containing cycloheximide (0.1 mg/ml), pelleted and resuspended in extraction buffer (20 mM Tris-HCl, pH 8.0, 140 mM KCl, 0.5 mM DTT, 5 mM MgCl2, 0.5 % nonidet-P40, 0.1 mg/ml cycloheximide, 10000 IU/ml dalteparin sodium) and incubated for 5 min on ice. All subsequent steps were carried out in the cold. After centrifugation for 10 min at 12000 x g, 0.4 ml supernatant was layered onto a 12 ml linear sucrose gradient (10 - 50% sucrose [w/v] in extraction buffer without nonidet-P40) and centrifuged at 4 Â°C in an SW40Ti rotor (Beckman) at 35000 rpm without brake for 120 min. The gradients were harvested and absorbance profiles at 254 nm recorded (type 11 optical unit/UA-6 detector, ISCO). 1 ml fractions were collected and supplied with 0.1 volume of 3 M sodium acetate (pH 5.2) and 1 volume of isopropanol for overnight precipitation at -20 Â°C. RNA was purified using Nucleospin RNAII tubes (Macherey & Nagel) following the manufacturerÂ´s protocol. For microarray analysis RNA from polysome fractions were pooled, precipitated by adding LiCl to a final concentration of 2 M and resolubilized in 15 Âµl of RNase-free water. RNA concentrations were determined and the samples were stored at -80 Â°C.

Project description:We sequenced pollen DNA from Col x Ler F1 hybrid plants using Nanopore long-read sequencing and identified crossover sites with COmapper. To purify pollen grains from F1 hybrid plants and extract their genomic DNA, whole inflorescences of hybrid plants were collected and transferred to ice-cold 10% (w/v) sucrose in a 50-ml tubes. Flowers were homogenized with a blender (Tefal, BL3051KR), filtered through 80-μm nylon mesh, and centrifuged at 350g for 10 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 10% (w/v) sucrose, filtered through a 40-μm cell strainer, and centrifuged at 100g for 10 min at 4°C. The purified pellet was frozen in liquid nitrogen and ground with a mortar and pestle. The pellet powder was resuspended in CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA). Proteinase K (NEB, P8107S) and RNase A (20 mg/ml, Roche, 10109142001) were added to a final concentration of 80 U/ml and 20 μg/ml, respectively. To check pollen disruption, a 1-μl sample was diluted in a 1:10 ratio with 10% (w/v) sucrose and observed under a stereomicroscope (Leica, M165 FC). Then, 500 μl of the resuspended pellet was transferred to a 1.5-ml tube, to which an equal volume of 25:24:1 (v/v/v) phenol:chloroform:isoamylalcohol (Sigma, 77617) was added, and the mixture was homogenized by gentle manual shaking. The tube was centrifuged at 15,000g for 10 min at 4°C, and 400 μl of supernatant was transferred into a new tube, to which a Nanobind disk (Nanobind® Plant Nuclei Kit RT, PacBio, 102-302-000) was added. The tube containing the Nanobind disk was gently mixed, 400 μl isopropanol was added, and the tube was slowly inverted and incubated for 2 h at room temperature on a rotator set at 7 rpm. A magnetic rack (DynaMagTM-2, Invitrogen, 12321D) was used to separate the disk and the supernatant, and the supernatant was removed. PW1 buffer in the Nanobind Plant Nuclei Kit was used for washing the disk twice and the supernatant was removed completely by centrifugation. Then, 150 μl EB+ buffer was added onto the disc and gently tabbed and incubated overnight at room temperature. The supernatant was transferred to a new 1.5-ml tube and stored at −20°C. The DNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853) and integrity checked by gel electrophoresis. Nine micrograms of gDNA from pollen was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).

Dataset Information

Shewanella oneidensis outer membrane and outer membrane vesicles

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets