M_Mesophilicum_Fe_Ln_limitation_08092024_K1_to_K33
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Methylobacterium Mesophilicum (ncbitaxon:39956)
SUBMITTER: 
Allegra Aron
PROVIDER: MSV000095569 | MassIVE | Fri Aug 09 18:09:00 BST 2024
REPOSITORIES: MassIVE
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Project description:Water electrospray is small charged droplets generated by a laboratory-designed prototype module. Extensive investigations have explored its physical properties and identified a potential non-chemical biocide against air-borne pathogens. However, since humans can inhale water electrospray particles through breath, humans' safety and potential toxicity warrant investigation. Hence, to offer insights into the effect of water electrospray on humans, we analyzed the immunopathological response to water electrospray using a nasal challenge mouse model. For this, we accessed the fundamental immune responses following the water electrospray intranasal challenge by comparing its effect with sodium hypochlorite, a biocidal agent with known toxicity. Our results indicate that water electrospray did not induce pathological immune reactions: challenged mice did not exhibit body weight loss and increased inflammatory cytokine production. Furthermore, histopathological analysis revealed that water electrospray did not damage the lung tissue, whereas sodium hypochlorite–treated mice showed significant lung tissue damage with signs of neutrophils and eosinophils infiltration. Finally, transcriptomic analysis on lung tissue confirmed the absence of pathological immune response in water electrospray-treated mice compared with sodium hypochlorite–treated mice. Together, we provide evidence that water electrospray is a safe technology for its use in disinfecting air-borne pathogens with little or no effect on immune system activation at the preclinical level.
2025-04-02 | GSE262972 | GEO
Project description:For gaining additional insights into the composition of the testicular proteome of the domestic pig (Sus scrofa domestica), we conducted 2DE-MS. Two-dimensional SDS PAGE was run on testicular lysates of three boars, with three gels per boar. Upon matching across gels, we arbitrarily selected protein spots for mass spectrometry analysis. Excised slices were vacuum dried and soaked with digestion buffer containing trypsin (0.01 μg/μl), followed by overnight incubation at 37°C in the same buffer without trypsin. Subsequently, peptides were extracted in solvents of increasing acetonitrile content, by sonication. Upon vacuum-centrifugation, peptides were reconstituted in 0.1% formic acid (FA). Following this, peptides were fractionated by reversed phase liquid chromatography (C18; buffer A: 0.1% FA dissolved in HPLC-H2O; buffer B: 0.1% FA, dissolved in CAN; flow-rate: 0.4 µL/min; gradient: 2-30% in 30 minutes). Eluted peptides were injected via an electrospray ionization interface into a Q-TOF mass spectrometer (one boar, Q TOF Ultima, Micromass/Waters, Manchester, UK) and an ion-trap mass spectrometer (two other boars, XCT ion-trap, Agilent Technologies, Waldbronn, Germany). We used ProteomeDiscoverer 2.4 (Thermo Fisher Scientific, San Jose, USA) for peptide and protein identification. Using Sequest HT, we searched peak lists (*.mgf) against the Sus scrofa reference proteome database (UniProt Proteome ID: UP000008227, 49,793 proteins).
2022-08-12 | PXD015649 | Pride
Project description:Positive electrospray ionization. Methylobacterium Extorquens. Iron and lantanide limitation. Collaboration with Penn State.
| MSV000095913 | MassIVE
Project description:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.
2021-07-12 | PXD025754 | Pride
Project description:TiO2 enriched phosphopeptides of Iloprost stimulated platelets were digested with trypsin and labeled with iTRAQ. Samples were analysed by nano LC-MS/MS on an LTQ OrbitrapVelos (Thermo Scientific) coupled to an Ultimate 3000 Liquid Chromatography system (Dionex). Peptides were concentrated on a self-packed 100 µm ID reversed-phase (RP) trapping column (Kinetex C18, 2 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) in 0.1% TFA followed by separation on a self-packed 75 μm ID RP column (Kinetex C18, 30 cm length, 2.6 µm particle size, 100 Å pore width; Phenomenex) using a binary gradient (solvent A: 0.1% FA and solvent B: 0.1% FA, 84% ACN) ranging from 3-45 % solvent B in 245 min at a flow rate of 230 nl/min. To minimize memory and carry-over effects from previous samples, an LC-wash program with high organic content was used prior to sample injection (Burkhart et al 2011). Survey scans were acquired in the Orbitrap with a resolution of 30,000 using the polysiloxane m/z 371.101236 as lock mass5 and MS/MS scans of the five most intense signals were acquired in the Orbitrap using HCD fragmentation (activation 0.1 ms, normalized collision energy of 50, isolation width of 1.5 m/z) with a resolution of 7,500. To compensate for elevated peptide charge states due to iTRAQ labels, a reaction tube containing 5 % ammonium water was placed in front of the ion source as described by Thingholm et. al. Database search and data processing: Raw data were processed with Proteome Discoverer 1.3 (Thermo Scientific) using Mascot (2.4) and Sequest for database search against the human Uniprot database (November, 4th 2010; 20,260 sequences) using the following search settings for both algorithms: i) trypsin with a maximum of two missed cleavages, (ii) carbamidomethylation of Cys (+57.0214 Da), peptide N-terminus iTRAQ, Lys iTRAQ (+144.102 Da) as fixed modifications and (iii) oxidation of Met (+15.9949 Da), Tyr iTRAQ (+144.102 Da) as well as phosphorylation of Ser/Thr/Tyr (+79.9663 Da) as variable modifications; (iv) MS and MS/MS tolerances of 10 ppm and 0.02 Da, respectively. A false discovery rate (FDR) of <1% was applied using the Peptide Validator node. Additionally, phosphorylation site localizations were validated by the phosphoRS node. Only phosphopeptide hits with <1% FDR of all validated peptides were considered for data interpretation and site assignment probabilities of >90 % (phosphoRS) were defined as confident. Data interpretation: To compensate for systematic variations or technical errors for each sample set, reporter areas were normalized based on modellized normal distribution with a +- 34% quantil. Therefore median iTRAQ ratios were calculated considering all unique <1% FDR peptides and normalization factors against the control sample were determined. For each peptide, all MS/MS spectra of one biological replicate were grouped in regard to modified site in protein and phosphoRS site localization (peptide groups) to determine the respective median ratios. Finally, for each peptide group, median values were calculated considering all three biological replicates. Peptides with ratios below 0.5 or above 2.0 were considered as regulated. Regulated phosphopeptides were searched against GPS 2.1 for kinase consensus site prediction. Values of high confidence (<2 % error) for PKA were taken into account.
2013-12-02 | PXD000242 | Pride
Project description:The experiment consists of M.hyp 232 cultures digests analyzed on two mass specs, LTQ Velos Pro and LTQ FT Ultra LTQ Velos Pro: Six cell culture replicates of M.hyo 232 were hydrophobically separated using tree detergents: Digitonin, Tween, and SDS. Each francion, including the insoluble pellet, was trypsin digested and then cleaned using an SCX trap follwed by a RP trap to remove detergents. Each fraction was sepated using a Dionex U3000 splitless nanoflow system operating at 333 nl per minute using a gradient of 2% ACN to 50% ACN in 4 hours. Eluate was analyzed using an LTQ Velos Pro mass spectrometer with 20 MS/MS scans of the 20 most intense peaks from each MS scan. Dynamic exclusion was enable for 3 minutes for each m/z with a repeat count of 1. LTQ FT Ultra: Two cell pellets for high resolution analysis were lysed and trypsin digested. Digested peptides were dried, resuspended in 20 mM KH2PO4, 20% ACN, pH 3 (Buffer A) in 2.5 µL and transferred to low retention vials in preparation for separation using an Ultimate 3000 configured for 2D-LC. Each sample was loaded at 15 µl/min onto an SCX microtrap for the first dimension of separation, involving SCX steps of Buffer A + 0, 5, 10, 15, 20, 25, 30, 40, 50, 100, 250, 500, and 1000 mM KCl. For the second dimension of separation, each eluted salt step was desalted with an inline peptide microtrap with 2% ACN, 0.1% FA at 5 µl/min. Once desalted, the microtrap was switched into line with a fritless nano column (75µm x ~10cm) containing C18 media (5µ, 200 Å Magic, Michrom). Peptides were eluted using a gradient of 2% to 36% ACN, 0.1% FA at 350 nl/min over 60 min and electrospray ionized for analysis using an LTQ FT Ultra mass spectrometer. A survey scan m/z 350-1750 was acquired in the FT ICR cell (Resolution = 100,000 at m/z 400, with an accumulation target value of 1,000,000 ions). Up to the 6 most abundant ions (>3,000 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 seconds. Analysis: X!tandem searches were performed using the Mycoplasma hyopneumoniae strain 232 reference protein set from NCBI. The only difference between the searches for the LTQ Velos and LTQ FT was the precursor mass tolenance being set to +-1500ppm and +-24ppm respectively. Decoy searches were performed and the data filtered at e-value <= 0.01 with single peptide proteins discarded. These results are included in the submission as two tab-delimited text files.
2018-01-10 | PXD000118 | Pride
Project description:Cyanophora paradoxa muroplasts were isolated on a Percoll gradient. Muroplasts were fractionated into soluble (stroma), envelope, and thylakoid proteins. Protein samples from envelope membranes, thylakoids, stroma and total muroplasts were separated by 12% SDS-PAGE. Each Protein lanewas cut into 10 slices of variable size to match similar protein amounts. The gel slices were washed and diced to 1mm3 cubes. Gel pieces were destained by two consecutive washes with a 50:50 mixture of 200 mM ammonium bicarbonate and acetonitrile (ACN) for 20 min at 37°C. Proteins were reduced with 10mM DTT for 45 min at 56°C, followed by alkylation with 55mM iodoacetamide for 45 min at room temperature in the dark. After reduction and alkylation, gel pieces were washed twice with 100 mM ammonium bicarbonate, dehydrated with 100% ACN for 10 min, and dried in a SpeedVac. Gel pieces were fully covered in 50 mM ammonium bicarbonate containing 10ng/ml trypsin and rehydrated at 4°C. The samples were incubated overnight at 37°C and peptides in the supernatant were pooled after successive extraction steps using 50 mM ammonium bicarbonate, 100% ACN and 2.5% formic acid (FA), 50% ACN. The samples were dried in a SpeedVac and reconstituted in 5% ACN, 0.1% FA prior to MS analysis. Each of the samples were analysed in duplicates. Tryptic peptides of the thylakoid, envelope and stroma fractions were loaded onto a fritless 100 μm capillary packed in-house with 10cm of ProntoSIL C18 ace-EPS, 3µm. A gradient of 5-80% (v/v) ACN in 0.1% (v/v) FA was passed over the column with a duration of 115 min. The eluted peptides were directly electrosprayed into the LTQ orbitrap XL MS at a flow rate of 250 nl min-1 and a spray voltage of 1.3 kV. The instrument was operated in a 4-step data-dependent positive mode to identify the top three most abundant ions in a high-resolution MS scan (400 to 2000 m/z), which were then selected for fragmentation. MudPIT analyses were performed on the muroplast samples using an in-house packed analytical column consisting of 10 cm ProntoSIL C18 ace-EPS, 3µm (Bischoff) and 2 cm of a strong cationic exchange (SCX) material, 3µm in combination with an 6-step salt pulse gradient (0, 50, 100, 150, 200 and 500 mM ammonium acetate). Each salt-step peptides were eluted from the SCX to the C18-phase of the analytical column and eluated with a 130- min reverse-phase solvent gradient (5–80% ACN containing 0.1% FA). The eluate was directly electrosprayed into the LTQ orbitrap XL MS which was operated as described before. All MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search the C. paradoxa nuclear and muroplast protein database with a total of 32,286 entries, assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the C. paradoxa database also assuming trypsin. Sequest and X! Tandem were searched with a fragment ion mass tolerance of 0,80 Da and a parent ion tolerance of 10,0 ppm. Oxidation of methionine and iodoacetamide derivatives of cysteine were specified in Sequest and X! Tandem as variable modifications. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95,0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99,0% probability and contained at least 2 identified peptides.
2012-10-30 | PXD000056 | Pride
Project description:Leaves of Brachypodium distachyon Bd21 were collected at three leaf stage. The total proteins were extracted and then phosphopeptides were enriched by using TiO2 microcolumns and phosphopeptides and their corresponding proteins were identified with LC-MS/MS and Maxquant software. In details, enriched phosphopeptides were separated on a self-packed C18 reverse phase column (75 μm I.D., 150 mm length) (Column Technology Inc., Fremont, CA), which directly connected the nano electrospray ion source to a LTQ-Orbitrap XL mass spectrometer. Pump flow was split to achieve a flow rate at 1 μL/min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of 0.1% FA (A) and 0.1% FA and 80% ACN (B). A five-step linear gradient of 5% to 30% B in 105 min, 35% to 90% B in 16 min, 90% B in 4 min, 90% to 2% B for 0.5 min and 2% B for 14.5 min was performed. The spray voltage was set to 2.0 kV and the temperature of the heated capillary was 240ºC. For data acquisition, a data-dependent top 10 MS/MS spectraper MS full scan in the Orbitrapanalyser was used. The raw files were processed with Maxquant (version 1.1.1.36) and searched against the NCBI Brachypodium protein database (26,035 entries in total) concatenated with a decoy of reversed sequences. The following parameters were used for database searches: cysteine carbamidomethylation was selected as a fixed modification; methionine oxidation, protein N-terminal acetylation, and phosphorylation on serine, threonine and tyrosine were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm, and fragment ion mass tolerance was 0.5 Da for MS/MS spectra.
2013-12-17 | PXD000340 | Pride
Project description:Oral submucous fibrosis (OSF) is one of the most common precancerous malignant orders (PMO) with high rates exacerbating into oral squamous cell carcinoma (OSCC), mostly occurred in patients with chewing betel-nut habits in Southeast Asia and Pacific region. However, the spatial characteristics and heteogeneities of tumor microenvironment (TME) in OSF-associated OSCC still remains unclear. Here, we characterized the spatiotemporal changes of OSF-associated OSCC at different malignant states by performing 10x Visium Spatial Transcriptomics (ST) sequencing and airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADESI-MSI) analysis.
2024-03-05 | GSE220978 | GEO
Project description:LC-MS/MS lipidomics data of Komagataella phaffii recorded on a Q Exactive HF with apex trigger in positive ion mode. Thermo Scientific Accucore C30 column (150 x 2.1 mm, 2.6 um) reverse phase column: A: ACN:H2O 6:4, 10 mM NH4Form, 0.1% FA, B: IPA:ACN 9:1, 10 mM NH4Form, 0.1% FA, 27 min gradient, follows Anal. Chem. 2018, 90 (11), 6494-6501.
2022-07-28 | MSV000090008 | MassIVE