Differential protein expression between Carfilzomib-sensitive (AMO1) and resistant (AMO1-CFZ) multiple myeloma cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): timsTOF Pro
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Blood Cell, Cell Culture
DISEASE(S): Multiple Myeloma
SUBMITTER: 
Animesh Sharma
LAB HEAD: Geir Slupphaug
PROVIDER: PXD045439 | Pride | 2024-06-07
REPOSITORIES: Pride
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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
Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.
2022-12-07 | PXD023141 | Pride
Project description:Proteomic analysis of pervanadate-induced tyrosine-phosphorylated proteins in hepatocellular carcinoma WRL 68 cells Protein tyrosine phosphorylation plays critical roles in modulating biological processes such as cellular proliferation, differentiation, migration, apoptosis and metabolism. To profile tyrosine phosphorylated (pTyr) proteins as well as search novel pTyr proteins as cross-talk points among different cellular pathways, we developed a rapid and efficient approach to identify cellular pTyr proteins and their complexes by a combination of subcellular proteomics approach with signal transduction strategies. Human hepatocytic cells from WRL68 cell line were treated with pervanadate (POV), subfractionated into four fractions and then subjected to immunoaffinity purification with anti-pTyr antibody. The eluted mixtures of the anti-pTyr purification were identified by LC-MS/MS. Subcellular fractionation and affinity purification of tyrosine-phosphorylated proteins: WRL68 cells were first grown to 80% confluence in MEM complete medium and then the medium replaced with serum free media. After 15 h, the cells were either untreated or stimulated with 0.1 mM pervanadate (1 mM sodium orthovanadate, 3 mM H2O2) for 10 min. 150-mm cultures of WRL 68 cells were rinsed twice with 4? PBS and then scraped from the dish in 750?l of hypotonic buffer (10 mM Tris, 1 mM NaF, 10 mM IAA, pH 7.5) containing a cocktail of protein inhibitors. After a 20-min incubation on ice, the cells were passed about five times through a 25-g needle. The resulting lysate was subjected to a 15min centrifugation at 1000 rpm at 4?, after which the pellet was resuspended in 250?l of hypotonic buffer and re-extracted by a second round of the trituration and centrifugation. The supernatants of the first and second spins were combined, adjusted to 0.25 M NaCl, and separated into cytosolic (supernatant) and membrane (pellet) fractions bycentrifugation at 19,000 rpm (43,000 g) for 90 min at 4?. All the pellets and total cell lysate were resuspended in RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% deoxycholic acid sodium) containing 1mM pervanadate with a cocktail of protein inhibitors with sonication aid. Cleared cell lysates were incubated overnight at 4? with 30?l monoclonal anti-phosphotyrosine-agrose (Sigma). Precipitated immune complexes were washed three times with 1×HNTG (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, 10% Glycerol, pH 7.5) and then eluted with 100 mM phenyl phosphate (Sigma) in lysis buffer at 4?. Enzyme digestion, mass spectrometry and protein identification: The sample was digested according to the published method. Chromatography was performed using a surveyor LC system (Thermo Finnigan,SanJose,CA) on C18 reverse phase column(RP, 180 µm x 150 mm, BioBasic® C18, 5 µm, Thermo Hypersil-Keystone). The pump flow rate is split 1:100 for a colum flow rate of 1.5 µL/min.The column effluent is directly electrosprayed using the orthogonal metal needle source without further splitting.Mobile phase A is 0.1% formic acid in water,and the B mobile phase is 0.1% formic acid in acetonitrile. The separation of peptides obtained by enzymatic digest of bile sample was achieved with a gradient of 2-80% B over 360 min.The column effluent from the reverse phase column was analyzed by LCQ Deca XP ion-trap mass spectrometer.The micro-electrospray interface uses a 30 µm metal needle that is orthogonal to the inlet of the LCQ.The mass spectrometer was set that one full MS scan was followed by three MS/MS scans on the three most intense ion from the MS spectrum with the following Dynamic Exclusion™ settings: repeat count, 1; repeat duration, 0.5 min; exclusion duration, 3.0 min. The acquired MS/MS spectra were automatically searched against protein database for human proteins (SWISS-PROT/TrEMBL) using the TurboSEQUEST program in the BioWorks™ 3.0 software suite. An accepted SEQUEST result had to have a ?Cn score of at least 0.1 (regardless of charge state) and Xcorr (one charge?1.5, two charges?2.0, three charges?2.5). Single peptides that alone identify a protein were manually validated after meeting the above criteria. Bioinformatics analysis: The pI and MW of the proteins were analyzed using ExPASy proteomics tools accessed from http://cn.expasy.org/tools/#proteome. The grand average hydropathicity (GRAVY) values were determined according to Kyte-Doolittle. The protein subcellular location annotation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/). The protein function family was categorized according to Gene Ontology (GO) annotation terms extracted by InterPro (http://www.ebi.ac.uk./interpro/). The annotation of protein phosphorylation was from SwissProt &TrEMBL protein database (http://us.expasy.org/sprot/) and PhosphoSite (http://www.phosphosite.org). The kinases were annotated according to human kinome. Keywords: other
2005-04-07 | GSE2408 | GEO
Project description:Human hepatoma HepG2 cells (American Type Culture Collection; HB-8065) were grown in 75 ml tissue cell culture flasks in Eagle's minimal essential medium (EMEM) augmented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO2. For treatment with fatty acids or analogues, the cells were seeded at a density of 2 × 106 cells in a T25-cm2 flask for 24 h. Each FA or analogue was added to the culture medium as a fatty acid-bovine serum albumin (BSA) complex (2.5:1, FA:BSA) to give the desired final concentration. The controls in these experiments were HepG2 cells with BSA alone. After 24 h treatment, the media was collected, cells were rinsed twice with PBS and cells were harvested for analysis. To each cell suspension prior to lipid extraction a standard mixture of 25 µg C15:0 PE, C17:0 PC and C71:1 TAG was added as standards. Extraction of lipids was performed according to the Folch method. For metabolomics analysis, the lipid extracts were resuspended in chloroform/methanol, 2:1 (v/v).All analyses were carried out using an Agilent 1200 Series HPLC, ACE 5 C8-300 column (2.1 x 100 mm) and linear gradient elution at a flow rate of 0.1 mL/min. Mobile phase A and B consisted of 0.1% formic acid; 10 mM ammonium acetate in H2O and 0.1% formic acid; 10 mM ammonium acetate in ACN/isopropanol (50/50; v/v), respectively. The injection volume was 4 µL. Separation of metabolites was achieved at the following gradient: T=0 min: 30% B; T=1 min: 30% B; T=25 min: 100% B; T=45 min: 100% B; T=47 min: 30% B; and T=60 min: 30% B (re-equilibration). The HPLC system was directly coupled to a Bruker Soalrix 70 Hybrid FTMS instrument equipped with electrospray ionization source (ESI) (Bruker Daltonics). The system was controlled by HyStar v.3.4.8.0 software. MS data was collected with resolving power of 78,000 (at m/z 400) in positive or negative mode under following conditions: a capillary voltage of (+/-) 4,500 V and an end plate offset of -500 V. The dry temperature was set at 180°C. Dry gas flow was maintained 4 L/min. Acquisition range was 244-1,800 m/z with 0.2 s ion accumulation time. LC-MS data was converted into mzXML format using CompassXport v. 3.0.6 and processed by mzMine v.2.10 [25] or XCMS data analysis software. Data processing involved mass detection, chromatographic peak detection and deconvolution, isotopic peaks grouping, normalization and peak alignment. Metabolite data were mean-centered and unit-variance scaled to remove the offsets and adjust the importance of high and low abundance metabolites to an equal level. Significantly altered metabolites were defined by a fold change (FC) >2 and p<0.05. Principal component analysis (PCA) and hierarchical clustering analysis (HCA) of signature metabolites altered in compounds treated cells compared to control were performed in the Metaboanalyst web portal (www.metaboanalyst.ca).
2015-09-09 | ST000241 | MetabolomicsWorkbench
Project description:The aim of the project was to purify Xenopus replisome from replicationg chromatin assembled in Xenopus laevis egg extract. To this end recombinant Cdc45-TEV-His10-FLAG5 was expressed in bacteria and purified. 4 ml of Xenopus laevis egg extract was activated into interphase and supplemented with 10 ng/µl of demembranated sperm DNA, 70 nM recombinant Cdc45, 40 µM aphidicolin, 5 mM caffeine and incubated at 23°C for 60 min. Chromatin was isolated in ANIB100 buffer (50 mM HEPES pH 7.6, 100 mM KOAc, 10 mM MgOAc, 2.5 mM Mg-ATP, 0.5 mM spermidine, 0.3 mM spermine, 1 µg/ml of each aprotinin, leupeptin and pepstatin, 25 mM β-glycerophosphate, 0.1 mM Na3VO4 and 10 mM 2-chloroacetamide) as described previously (Gillespie, Gambus et al. 2012). Chromatin pellets re-suspended in ANIB100 containing 20% sucrose. Protein complexes were released from chromatin by digestion with 4 U/µl benzonase nuclease (E1014-25KU, Sigma) and sonicated for 5 min using a sonicator with settings: 30 sec on, 30 sec off, low power (Bioruptor Diagenode UCD-200). Immunoprecipitation was performed using 100 µl of anti-FLAG M2 magnetic beads (Sigma-Aldrich). Before elution the sample was washed four times with 1 ml of ANIB100 20% sucrose, ANIB100 20% sucrose 0.1% Triton X-100, ANIB100 and elution buffer (25 mM HEPES pH 7.5, 100 mM KAc, 5 mM MgAc, 1 mM ATP and 0.02% NP-40), respectively. The sample was eluted adding 250 µM 3xFLAG peptide (Stratech) to 200 µl of elution buffer and a small proportion of it analysed by mass spectrometry.
2023-10-27 | PXD044422 | Pride
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).
2012-12-10 | PXD000004 | Pride
Project description:<p>The sample to be tested was fully ground into powder in the grinder, 50 mg of the sample was freeze-dried, 1000 μL of the extraction solution containing the inner target was added (methanol:acetonitrile:water, 2:2:1, v/v/v, internal standard concentration 20 mg/L) and vortex mixed for 30 s; then add the steel ball to the 45 Hz grinder for 10 min, ultrasonic 10 min; the sample to be tested is obtained after filtration. When detecting metabolites, metabolite determination was performed based on the LC-MS system, which mainly consists of Waters Acquity I-Class PLUS ultra-high performance liquid tandem and Waters Xevo G2-XS QTof high-resolution mass spectrometer. Meanwhile, the Waters Acquity UPLC HSS T3 column (1.8 um 2.1 x 100 mm) was used as the chromatographic column. Positive and negative ion modes were used to determine the metabolites. Mobile phase A: 0.1% formic acid aqueous solution; Mobile phase B: 0.1% acetonitrile formate. The mobile phase conditions of liquid chromatography were as follows: the flow rate was 400 μL/min, 0.0 min: 98% flow A, 2% flow B; 0.25 min: 98% flow A, 2% flow B, 10.0 min: 2% flow A, 98% flow B; 13.0 min: 2% flow A, 98% flow B; 13.1 min: 98% flow A, 2% flow B; 15.0 min: 98% flow A, 2% flow B. MSe mode controlled by acquisition software (MassLynx v4.2, Waters) was used for primary and secondary mass spectrum data acquisition. ESI ion source parameters are as follows: Capillary voltage, 2500 V (positive ion mode) or -2000 V (negative ion mode); Cone hole voltage, 30 V; Ion source temperature, 100 °C; Desolvent temperature, 500 °C; Air flow rate, 50 L/h; Desolvent gas flow rate, 800 L/h; Plastic-nucleus ratio (m/z) collection range 50-1200 m/z. In the qualitative and quantitative analysis of metabolites, the original data collected by MassLynx v4.2 were processed by the Progenesis QI software for peak extraction, peak alignment, and other data, and the metabolites were identified based on the Progenesis QI software online METLIN database. Then, based on the results of the total score, MS2 score, and mass error (ppm), the metabolites were qualitatively determined.</p>
2024-07-12 | MTBLS10002 | MetaboLights
Project description:To probe the binding sites of MeCbl on GSDME, 0.4 μM recombinant human GSDME (CSB-EP006766HU) purchased from CUSABIO (Wuhan, China) was incubated with 10 μM MeCbl in 100 μL buffer (20 mM HEPES, 150 mM NaCl and 50 μM DPA) at room temperature for 2 h and protected from light. 0.4 μM recombinant human GSDME was incubated with ddH2O as negative control, or with HOCbl for binding sites analysis between GSDME and cobalamin. All groups were equally prepared ten incubation samples and mixed for further analysis. Trypsin and Glu C were then added in the mixture overnight, and formic acid was added to adjust the pH down to three to terminate the enzyme digestion. Activated the C18 desalting column with 100 μL 100% acetonitrile, then centrifuged at 400 g for 3 min. Added 100 μL of 0.1% formic acid and centrifuged at 400 g for 3 min for column equilibration. Replaced with new EP tube, added samples to column, and centrifuged at 400 g for 3 min. Washed the column twice with 100 μL of 0.1% formic acid and centrifuged at 400 g for 3 min, washed once again with 100 μL water at pH10. The eluents were collected with 70% acetonitrile, each sample was lyophilized and stored at -80°C until further mass spectrometry analysis. Prepared the mobile phase solution, A (0.1% (v/v) formic acid in ddH2O), B (0.1% (v/v) formic acid and 80% acetonitrile in ddH2O), the lyophilized samples were dissolved with 10 µL solution A, then centrifuged at 14000 g for 20 min at 4°C, 400 ng supernatant of the samples were injected onto a Thermo Scientific™ UltiMate™ 3000 UHPLC coupled to a timsTOF HT mass spectrometer (BRUKER). Peptides were then separated over a 100 μm ⅹ15 cm ReproSil-Pur C18-AQ 1.5-µm silica beads (Beijing Qinglian Biotech Co.,Ltd, Beijing, China), at a flow rate of 300 nL/min using a gradient: 0-4 min (5-10% B), 4-46 min (10-24% B), 46-53 min (36% B), 53-54 min (95% B), 54-60 (95% B). The timsTOF HT mass spectrometer was used for liquid quality detection with Captive Spray source, and the mass spectrum was collected in DDA mode. The scanning range of the mass spectrum was m/z 100-1700, the primary mass spectrum resolution was set to 60,000 (1222 m/z), and the cumulative time was set to 100 ms in the TIMS tunnel. The capillary voltage is set at 1.6 kV and the mobility is 0.6-1.6 cm2/(V). The total cycle time was 1.1s with 10 PASEF cycles. The mass spectrometry raw data was processed by FragPipe to search against the target protein database. The precursor ion mass tolerance was ±15 ppm; the fragment mass tolerance was ±0.02 Da. The static modification was carbamidomethylation of cysteine; the dynamic modifications were oxidation of methionine, acetylation of N-termini of peptides and C63H91CoN13O14P or C62H88CoN13O14P of cysteine; a maximum of two missed cleavages were allowed.
2024-11-11 | PXD057768 |
Project description:MIADB: a cumulative collection of 172 tandem mass spectrometry (MS/MS) of a vast array of monoterpene indole alkaloids. Samples were analyzed using an Agilent LC-MS system composed of an Agilent 1260 Infinity HPLC coupled to an Agilent 6530 ESI-Q-TOF-MS operating in positive mode. A Sunfire analytical C18 column (150 × 2.1 mm; i.d. 3.5 μm, Waters) was used, with a flow rate of 250 μL/min and a linear gradient from 5% B (A: H2O + 0.1% formic acid, B: MeOH) to 100% B over 30 min. ESI conditions were set with the capillary temperature at 320 °C, source voltage at 3.5 kV, and a sheath gas flow rate of 10 L/min. The divert valve was set to waste for the first 3 min. There were four scan events: positive MS, window from m/z 100−1200, then three data-dependent MS/MS scans of the first, second, and third most intense ions from the first scan event. MS/MS settings were three fixed collision energies (30, 50, and 70 eV), default charge of 1, minimum intensity of 5000 counts, and isolation width of m/z 2. In the positive-ion mode, purine C5H4N4 [M + H]+ ion (m/z 121.050873) and the hexakis(1H,1H,3H-tetrafluoropropoxy)-phosphazene C18H18F24N3O6P3 [M + H]+ ion (m/z 922.009 798) were used as internal lock masses. Full scans were acquired at a resolution of 11 000 (at m/z 922). A permanent MS/MS exclusion list criterion was set to prevent oversampling of the internal calibrant.
2018-12-03 | MTBLS142 | MetaboLights
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