Si11 - standards mixture to test annotation of ISF features
Ontology highlight
ABSTRACT:
INSTRUMENT(S): maXis II
ORGANISM(S): Pure Standards Mixture
SUBMITTER: 
Raimo Franke
PROVIDER: MSV000095525 | MassIVE | Tue Aug 06 07:14:00 BST 2024
REPOSITORIES: MassIVE
Similar Datasets
Project description:A mixture of 11 compounds (2-methoxybenzoic acid, biochanin A, trans-ferulic acid, 3-indoleacetonitrile, indole-3-carboxaldehyd, kaempferol, kinetin, p-coumaric acid, L-(+)-alpha-phenylglycine, phloridzin dihydrate, rutin trihydrate) each at a concentration of 20 µM was prepared in a 1:1 mixture of acetonitrile and water.
1 µl of the Si11-mixture was analyzed by reversed phase ultrahigh-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry multiple times. The sample was separated using ultra high-performance liquid chromatography, performed on a Dionex Ultimate 3000 UPLC system (Thermo Fisher Scientific, Waltham, MA) using a 150 by 2.1 mm Kinetex C18 column with 1.7-mm particle size (Phenomenex, Aschaffenburg, Germany) column with a flow rate of 300 ml/min. Gradient elution with water with 0.1% (vol/vol) formic acid as eluent A and acetonitrile with 0.1% (vol/vol) formic acid as eluent B was run as follows: 1% B for t = 0 min to t = 2 min, linear gradient from 1% B to 100% B from t = 2 min to t = 20 min, hold 100% B until t = 25 min, and linear gradient from 100% B to 1% B from t = 25 min to t = 30 min. The sample was analyzed by positive mode electrospray ionization quadrupole time-of-flight mass spectrometry on a maXis HD QTOF (Bruker, Bremen, Germany) using data-dependent MS/MS by collision-induced dissociation of the three most abundant ions in each scan, making use of Bruker’s “Smart Exclusion” functionality to minimize multiple fragmentation of the same ion. Accurate masses were obtained by internal calibration using an ion cluster of sodium formate and lock mass calibration.
2024-08-06 | MSV000095525 | GNPS
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Project description:Ammonium hydroxide and pyrrolidine eluents were dried (SpeedVac) and reconstituted in 25 ul sample loading buffer (0.1% TFA/2% acetonitrile). Eluent from phosphotyrosine immunoprecipitation was dried and reconstituted in 35 ul of the loading buffer. An LTQ Orbitrap XL (ThermoFisher) in-line with a Paradigm MS2 HPLC (Michrom bioresources) was employed for acquiring high-resolution MS and MS/MS data. Ten microliters of the phospho-enriched peptides were loaded onto a sample trap (Captrap, Bruker-Michrom) in-line with a nano-capillary column (Picofrit, 75 um i.d.x 15 um tip, New Objective) packed in-house with 10 cm of MAGIC AQ C18 reverse phase material (Michrom Bioresource). Two different gradient programs, one each for MOAC and phosphotyrosine immunoprecipitation samples, were used for peptide elution. For MOAC samples, a gradient of 5-40% buffer B (95% acetonitrile/1% acetic acid) in 135 min and 5 min wash with 100% buffer B followed by 30 min of re-equilibration with buffer A (2% acetonitrile/1% acetic acid) was used. For phosphotyrosine immunoprecipitation samples, which were a much less complex mixture of peptides, 5-40% gradient with buffer B was achieved in 75 min followed by 5 min wash with buffer B and 30 min re-equilibration. Flow rate was ~0.3 ul/min. Peptides were directly introduced into the mass spectrometer using a nano-spray source. Orbitrap was set to collect 1 MS scan between 400-2000 m/z (resolution of 30,000 @ 400 m/z) in orbitrap followed by data dependent CID spectra on top 9 ions in LTQ (normalized collision energy ~35%). Dynamic exclusion was set to 2 MS/MS acquisitions followed by exclusion of the same precursor ion for 2 min. Maximum ion injection times were set to 300 ms for MS and 100 ms for MS/MS. Automatic Gain Control (AGC) was set to 1xe6 for MS and 5000 for MS/MS. Charge state screening was enabled to discard +1 and unassigned charge states. Technical duplicate data for each of the MOAC elutions (ammonium hydroxide and pyrrolidine) and triplicate data for the phosphotyrosine immunoprecipitation samples were acquired. RAW files were converted to mzXML using msconvert and searched against the Swissprot Human protein database (2013-Jan release) appended with common proteomics contaminants and reverse sequences as decoys. Searches were performed with X!Tandem (version 2010.10.01.1) using the k-score plugin. For all searches the following search parameters were used: Parent monoisotopic mass error of 50 ppm; fragment ion error of 0.8 Daltons; allowing for up to 2 missed tryptic cleavages. Variable modifications were oxidation of Methionine (+15.9949@M), carbamidomethylation of Cysteine (+57.0214@C), and phosphorylation of Serine, Threonine, and Tyrosine (+79.9663@[STY]). The search results were then post-processed using PeptideProphet and ProteinProphet.
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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.
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Project description:LTQ folder:
It cointains 16 folders (8 CTRL and 8 siRNA TTN; 4 biological replicates x 2 technical replicates per condition)
Q EXA folder:
It cointains 16 folders (8 CTRL and 8 siRNA TTN; 4 biological replicates x 2 technical replicates per condition)
LCQ DECA XP folder:
It cointains 2 folders corresponding to the analysis of proteins from pooled raft fractions.
About LTQ and LCQ DECA XP, peptide mixtures were first separated by means of ion-exchange chromatography using seven steps of increasing ammonium chloride concentration (0, 50, 100, 150, 200, 300, and 600 mM). Each salt step was directly loaded onto the reversed phase column and separated with an acetonitrile gradient: eluent A, 0.1% formic acid in water; eluent B, 0.1% formic acid in acetonitrile; the gradient profile was 5% B for 3 min followed by 5 to 50% B within 40 min.
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2015-05-26 | PXD001592 | Pride
Project description:Human primary macrophages were infected with influenza A virus (H3N2/Udorn strain, HA 256) for 6 hrs or left untreated. Cells were collected and lysed with HEPES lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 % NP-40, pH 7.4) including protease and phosphatase inhibitor cocktails. The cell lysates were centrifuged and the supernatant was collected and the protein content was measured with Bio-Rad DC™ protein assay (Bio-Rad). The proteins were reduced, alkylated, and enzymatically digested in-solution with trypsin. The digestion was stopped by adding FA (final c = 1 %). The samples were centrifuged 9168 x g for 10 min and desalted with Sep-Pak Vac RP C18 cartridges (Waters, MA, USA), following fractionation by strong cation exchange chromatography (SCX). The peptides were separated on a 200 x 4.6 mm, 5 μm, 200 Å PolySULFOETHYL A™ column (PolyLC, USA). The fractions were vacuum centrifuged and desalted as before, following phosphopeptide-enrichment with PHOS-Select™ Iron Affinity Gel (Sigma Aldrich, MO, USA). LC-MS/MS was performed with a Q Exactive hybrid quadrupole-orbitrap tandem mass spectrometer coupled to an EASY-nLC 1000 nanoflow liquid chromatograph (Thermo Fisher Scientific). A 100 μm x 3 cm trap column and a 75 μm x 15 cm analytical column were in-house packed with Magic C18AQ resin (200 Å, 5 μm; Michrom Bioresources). The mobile phases were 2% acetonitrile, 0.2% formic acid (A) and 95% acetonitrile, 0.2% formic acid (B). LC gradient elution condition was 2% B (0 min), 20% B (70 min), 40% B (100 min), and then 100% B (105-110 min), with a flow rate of 300 nl/min. Data dependent acquisition was performed in positive ion mode. MS spectra were acquired from m/z 300 to m/z 2000 at a resolution of 70,000 at m/z 200 with a target value of 1,000,000 and maximum injection time of 120 ms. The 10 most abundant precursor ions of which charge states were 2+ or higher were selected for higher energy collisional dissociation (HCD) with an isolation window of 2 and normalized collision energy of 30. MS/MS spectra were acquired at a resolution of 17,500 at m/z 200 with a target value of 50,000, maximum injection time of 250 ms, and the lowest mass fixed at m/z 100. Dynamic exclusion duration was 30 s.
2016-08-08 | PXD001620 | 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