A comparison of acetic and formic acid in single H358 cells analyzed by diaPASEF
Ontology highlight
ABSTRACT:
INSTRUMENT(S): timsTOF SCP
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Benjamin C Orsburn
PROVIDER: MSV000094565 | MassIVE | Thu Apr 18 09:00:00 BST 2024
SECONDARY ACCESSION(S): PXD051562
REPOSITORIES: MassIVE
Similar Datasets
Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.
2014-02-14 | PXD000354 | Pride
Project description:Frozen PBMC from infants 7 days before randomization (PBMC from two infant were 28 days after randomization) were stimulated in RPMI with 10% Feotal calf serum (FCS) at 5 million/ml with 1 million CFU/ml BCG (SII) or left unstimulated in a total volumn of 200ul/well in 96-well plates. Cells were incubated for 12 hours (37C, 5% CO2). Plates were centrifuged and 200ul supernatant was removed without disrupting the pellet and transferred into clean U-bottom plates. The plates were centrifuged again, the supernatant were flicked-out and the cells were resuspended in 200ul RLT buffer with beta-mercaptoethanol (10ul/ml). The samples were mixed with the buffer to lyse the cells and the plates were sealed and stored at -20C. At the time of RNA-extraction, plates were thawed at 37% for 10 mins. RNA extraction was performed using the Qiagen RNeasy kit following manufacturer's instructions (including optional DNase digestion) and RNA was eluted twice, 30 ul per elution. RNA was quantified by nanodrop and samples were stored at -80C.
2022-05-24 | GSE203395 | GEO
Project description:This experiment tests the effect of individual mutations on the metabolome of Arabidopsis. The standardized growth conditions are as follows: 1. Seeds (between 14 and 16) are sown on media in 100 x 100 x 15mm square Falcon Petri Dishes (Fisher Scientific, catalogue #08-757-11A). Seeds were arranged on the plates in a single horizontal line at the 1-cm mark from the top of the plate. 2. Each plate contains between 20 and 25-ml of sterile MS media containing 0.1% (w/v) sucrose. 3. Prior to sowing, seeds were sterilized by treating for 1-minute at room temperature with a 300-l solution of 50% (v/v) ethanol, this solution was removed and replaced with a 300-l solution consisting of 1% (v/v) Tween 20 (Fischer BioReagents, catalogue #BP33750), and 50% (v/v) bleach solution (Clorox), and incubated at room temperature for 10-minutes. The seeds were then washed with three changes of 0.3-ml of sterile water. 4. Upon sowing with seeds, the plates were wrapped with Micropore tape (3M Health Care, catalogue #1530-0), and then stored horizontally for 4-days at 4 °C, with illumination of 1 mol/m2. 5. On the 5th day, plates were moved to the growth room, and held in a vertical position in Plexi-glass holders for 17-days this growth room condition is labeled in Table I. 6. On 18th day Petri plates were opened and the aerial portions of these plants were harvested immediately upon plate opening. 7. Upon harvesting, plant material was quenched by immersion in liquid nitrogen and stored at 70 °C.
2013-05-20 | ST000013 | MetabolomicsWorkbench
Project description:This experiment tests the consequence of a mutation at the FatB gene (At1g08510) in the wound-response of Arabidopsis. The FatB mutant allele (fatb KD J. Ohlrogge (Plant Cell 2003, Vol 15, 1020-1033)) was obtained from Dr. Katayonn Dehesh, University of California, Davis, Davis, CA. This allele is in the Ws background.The standardized growth conditions are as follows: 1. Seeds (between 14 and 16) are sown on media in 100 x 100 x 15mm square Falcon Petri Dishes (Fisher Scientific, catalogue #08-757-11A). Seeds were arranged on the plates in a single horizontal line at the 1-cm mark from the top of the plate.2. Each plate contains between 20 and 25-ml of sterile MS media containing 0.1% (w/v) sucrose.3. Prior to sowing, seeds were sterilized by treating for 1 minute at room temperature with a 300-l solution of 50% (v/v) ethanol, this solution was removed and replaced with a 300-l solution consisting of 1% (v/v) Tween 20 (Fischer BioReagents, catalogue #BP33750), and 50% (v/v) bleach solution (Clorox), and incubated at room temperature for 10-minutes. The seeds were then washed with three changes of 0.3-ml of sterile water.
2013-01-15 | ST000001 | MetabolomicsWorkbench
Project description:We reported that the microRNA profiling of exosome isolated from 58 cerebrospinal fluid (CSF) of 38 medulloblastoma (MBL) patient with or without letomeningeal metastases (LM). CSF exosome was precipitated using Exo2DTM for RNA assay (EXOSOME plus, Suwon, South Korea) according to the manufacture’s protocol (Kim, et al. 2017). 2 ml of CSF were added to Exo2DTM reagent B following mixing by inverting, and incubated at room temperature for 30 min. The supernatant was discarded and the exosome containing pellet was resuspended in 100 ul of PBS to further RNA isolation. Total RNA was extracted from the CSF exosome fraction using a microRNA purification kit (Norgen Biotek corp., Thorold, Canada) according to the manufacture’s protocol. Briefly, samples were mixed with lysis buffer, and the lysate was added to absolute ethanol. The lysate was applied up to the column and centrifuged at 8,000 rpm for 1 min. The column was washed with wash solution and centrifuged at 14,000 rpm for 1 min. Finally, the column was applied to 20 ul of elution solution and centrifuged at 14,000 rpm for 2 min, and the flow-through was stored at –80 ℃ for storage. RNA precipitates were quantified and qualified with Bioanalyzer Pico 6000 Chip (Agilent Technologies, Santa Clara, CA). The 10 ng of total RNA was provided to the cDNA library generation using SMARTer smRNA-Seq kit (Illumina Inc., San Diego, CA), according to the user manual. The cDNA library was subjected to sequencing using Truseq SBS Kit and HiSeq 2500 System (Illumina Inc., San Diego, CA), according to user guide.
2020-09-15 | GSE157916 | GEO
Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.
2021-07-08 | PXD019017 | Pride
Project description:A control group consisting of nine C57/BL/6J mice was maintained on a normal diet consisting of Purina Rodent Chow (Harlan Teklad, catalog #5008, 6.5% fat, 49% carbohydrate, 23% protein, 3.5 kcal/ g). The experimental group consisted of nine mice grown on a specialty high-fat diet (Harlan Teklad, catalog #TD88137, 42.16% fat, 42.81% carbohydrate, 15.02% protein, 4.53 kcal/ g). Mice were maintained for ten weeks on their respective diets in a controlled environment with standard light and feeding schedule. Two days before tissue harvest, the experimental group on the high-fat diet was divided into one group of five and one group of four mice. The first group continued on the high-fat diet for the final two days, while the second group fasted. Mouse weights were recorded two days prior, and the day of tissue harvest. At the end of the fasting period liver tissue samples were harvested from each mouse. RNA was purified from the liver tissue samples. Each tissue was homogenized in 1 mL of STAT-60 (Tel-Test, Inc., Friendswood, TX) and the homogenate was mixed with 200 uL of chloroform. The aqueous phase was removed, and mixed with 500 uL of isopropanol to precipitate the RNA. The resulting RNA pellet was washed with 500 uL of 70% ethanol and allowed to air dry. The pellet was dissolved in RNase free water (Ambion, TX), quantified by UV spectroscopy, and stored at -80C. Total RNA control samples (Universal Mouse Reference RNA, catalog # 740100, Stratagene) were labeled using Cy3 dCTP (Perkin-Elmer), and liver total RNA experimental samples were labeled using Cy5 dCTP (Perkin-Elmer) during reverse transcription. For reverse transcription 10 ug of total RNA was mixed with 2 uL of 10X Cy-labeled dCTP, 2 uL of 100 mM DTT (Invitrogen), 4 uL of 5X First Strand Buffer (Invitrogen), 2 uL of a dNTP mixture containing 5 mM dGTP, dATP, dTTP and 2 mM dCTP (Invitrogen), 2 uL of 0.5 mg/ mL oligo-dT18-20 (Invitrogen), and 2 uL of Superscript II reverse transcriptase (Invitrogen). The control RNA samples were additionally mixed with 2 uL of random hexamers. The reaction mixtures were incubated at 42 C for two hours. After the reverse transcription reactions, the RNA templates were degraded by addition of 2 uL of 1 N NaOH and incubation at 65 C for 10 minutes. The alkaline conditions were neutralized by addition of 2 uL of 1 N HCl. Liver cDNA samples were then mixed with a control cDNA sample, and the mixtures were cleaned using a QIAquick Nucleotide Removal kit (Qiagen) as described by the manufacturer's instructions. The cleaned samples were concentrated in a speed vacufuge (Eppendorf) for 20 minutes at 65 C, and were dissolved in 32 uL of prewarmed hybridization buffer (Clontech Laboratories), quantified, and hybridized to prepared oligonucleotide microarrays. Hybridization occurred overnight at 55 C using Corning hybridization chambers. The following day the arrays were washed in Glass Hybridization Wash Solution (Clontech Laboratories), then in 1X SSC, and finally in 0.1X SSC. The arrays were dried by centrifugation and scanned using a GenePix 4000B scanner (Axon Instruments). The resulting data was downloaded and formatted in Excel (Microsoft), and analyzed using Matlab (The MathWorks, Inc.). Microarrays were prepared using GAPS glass slides (Corning), and a Virtek arrayer (Bio-Rad). Arrays contained 16,847 features, printed from a synthesized oligonucleotide mouse library (Operon). The library was suspended in 3X SSC, centrifuged, and loaded into the arrayer. Arrays were printed at approximately 50% humidity. Once printed, the probes were crosslinked using a UV Stratalinker (Stratagene). The slides were then blocked for 15 minutes in a 250 mL mixture containing 15.7 g/ L succinic anhydride (Fluka AG), 239 mL 1-methyl-2-pyrrolidinone (EM Science), and 10.7 mL of 1 M boric acid (Fisher), pH 8.0. They were cleaned by rinsing twice in 250 mL of milli-Q water, followed by 250 mL of 95% ethanol (Pharmco Products). After blocking, they were dried using filtered air and stored in the dark at room temperature. In this experiment the gene expression data of the control mice is given by samples GSM6592, GSM6593, GSM6594, and GSM6595. The gene expression data of the high-fat mice is given by samples GSM6599, GSM6600, GSM6601, and GSM6602. The gene expression data of the fasted mice is given by samples GSM6596, GSM6597, and GSM6598. Keywords = mouse, liver, energy homeostasis, gene expression, microarrays Keywords: repeat sample
2003-05-16 | GSE432 | GEO
Project description:ABSTRACT FOR BOTH PLASMA AND PLATELET-LYSATE In this article we describe the involvement of exchange factor activated by cAMP 1 (Epac1) in hemostasis and platelet activation. We have used plasma and platelet-lysate from EPAC1 knockout mice and wild-type control mice in label-free proteomics with an Orbitrap Velos Pro. Blood was obtained from wild-type and Epac1-/- mice euthanized with CO2. Approximately 1000 ul was drawn from the left ventricle into a 2 ml syringe, containing 100 ul ACD and 200 ul modified Tyrode`s buffer. The blood was centrifuged at 200g for 5 minutes at room temperature, and the resulting platelet-rich plasma (PRP) centrifuged at 700g for another 10 minutes in the presence of 10 ul ACD. The resulting platelet-poor plasma (PPP) was transferred to Eppendorf tubes and the pelleted platelets resuspended in modified Tyrode`s buffer and adjusted to 2.5 x 108 platelets/ml. Platelets were allowed to rest at room temperature before experimentation. For proteomics analysis, platelets were further purified by size-exclusion through Sepharose CL-2B gel (Pharmacia Biotec, Sweden) as previously described by Jensen et al. in Blood 2004; 104. Plasma was crude or depleted for Albumin prior to trypsination and LCMS with quantification using Progenesis LCMS. The platelet-lysates were fractioned on SDS-PAGE prior to trypsination and LC-MS analysis with MaxQuant quantification. Label-free protein quantification for plasma The software Progenesis LC-MS® Ver 2.7 (Nonlinear Dynamics Ltd, Newcastle, UK) was used for label-free quantification and comparison of LC-MS proteomics data based on the volume, m/z and retention time of the MS1 features (peptides). In Progenesis, the LC-MS runs were automatically aligned, and only features with charges between +2 to +7 and containing associated MSMS spectra were accepted for export as an mgf file for identification. The mgf file was search against the human SwissProt Mus musculus database (version September 2012) using SearchGUI Ver 1.8.9. The search criteria were: trypsin as the protease with no miss-cleavages accepted, fixed carbamidomethylation on cystein, variable oxidation on methionine, precursor mass tolerance of 10 ppm, fragment mass tolerance of 0.7 and OMSSA as the search engine. The search result and associated spectra were combined and assigned to proteins in Peptide Shaker Ver 0.17.3 (http://peptideshaker.googlecode.com) at 1% FDR. The results were exported from PeptideShaker as validated PSMs in a Phenyx format, and imported back into Progenesis. The protein abundances reported from Progenesis were based on the sum of the normalized abundance of the unique identified petides. The proteomics raw files and PRIDE XML identification files were uploaded to PRIDE using ProteomeXchange ver 1.0.4, and the projects are public available. Two PRIDE XML files were generated using PeptideShaker, one for crude plasma and one for albumin-depleted plasma.
2017-07-25 | PXD000282 | Pride
Project description:Immunoaffinity Purifications of Human Argonaute Twelve hours after transfections with mock or with miR-124, we washed each 15-cm plate once with phosphate-buffered saline (usually two plates were used per IP), then added 1 ml of 4 C lysis buffer (150 mM KCl, 25 mM Tris-HCl [pH 7.4], 5 mM Na-EDTA [pH 8.0], 0.5% Nonidet P-40, 0.5 mM DTT, 10 ul protease inhibitor cocktail [Pierce Cat# 78437], 100 U/ml SUPERaseIn [Ambion Cat# AM2694]). Following a 30-min incubation at 4 C, we scraped the plates, combined the lysates, and then spun them at 4 C for 30 min at 14,000 RPM in a microcentrifuge. We collected the supernatant and filtered it through a 0.45-um syringe filter. We froze an aliquot of lysate in liquid nitrogen for reference RNA isolation. We then added 0.22 mg/ml heparin to the lysate. We mixed the lysate with 2.5 mg of Dynal m-280 streptavidin beads (250 ul from original storage solution) coupled to biotinylated 4F9 ago antibody (~12.5 ug), which we equilibrated immediately prior to use by washing twice with 1 ml of lysis buffer. We incubated the beads with the lysate for 2 h at 4 C and then washed the beads twice with 1.25 ml of ice-cold lysis buffer for 5-min each. Five percent of the beads were frozen for SDS PAGE analysis after the second wash. RNA was extracted directly from the remaining beads using lysis buffer from Invitrogen's Micro-to-Midi kit (Invitrogen Cat# 12183-018). We purified RNA from the lysate and RNA extracted from the beads with the Micro-to-Midi kit as per vender's instructions, except that the percentage isopropanol used for binding to the column was 70%, instead of 33%, to promote the binding of small RNAs. RNA was amplified with ambion kit 1755. RNA from total cell lysate was labeled with cy3 and IPd RNA was labeled with cy5
2009-11-10 | GSE18834 | GEO
Project description:Total RNA was extracted from 400 ul of serum with the miRNeasy Serum/Plasma advanced Kit (Qiagen) and quantified using the Qubit microRNA assay kit (Thermo Fisher Scientific). cDNA templates were prepared using the TaqMan Advanced miRNA cDNA Synthesis Kit (Thermo Fisher Scientific), starting from 10 ng of RNA. RT-qPCR carried out on a QuantStudio 12K Flex (Applied Biosystems) using the TaqMan OpenArray miRNA panel.
2024-10-30 | GSE279435 | GEO