Mouse Neuroblastoma Cells Proteome by LC-MS/MS
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Ling Zhao
PROVIDER: PXD044493 | iProX | Wed Aug 09 00:00:00 BST 2023
REPOSITORIES: iProX
Publications
Lyssavirus M protein degrades neuronal microtubules by reprogramming mitochondrial metabolism.
Yuan Yueming Y Fang An A Wang Haoran H Wang Caiqian C Sui Baokun B Zhao Jianqing J Fu Zhen F ZF Zhou Ming M Zhao Ling L
mBio 20240213 3
Infection with neurotropic viruses may result in changes in host behavior, which are closely associated with degenerative changes in neurons. The lyssavirus genus comprises highly neurotropic viruses, including the rabies virus (RABV), which has been shown to induce degenerative changes in neurons, marked by the self-destruction of axons. The underlying mechanism by which the RABV degrades neuronal cytoskeletal proteins remains incomplete. In this study, we show that infection with RABV or overe ...[more]
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2013-05-28 | PXD000195 | 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: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:The chromatin fraction was isolated from 5x107 HeLa cells. Chromatin was digested in 100 mL of MNase (40 units/ mL ) reaction buffer for 3 min at 37 °C in a thermomixer (1,400 rpm). After addition of 10 L EGTA (25mM) to inactivate MNase, soluble digested chromatin was collected by 13,000 rpm centrifuge for 5 min. 10 mg of Pol II antibody was conjugated to magnetic protein A beads. The supernatant was diluted with 400 mL of NET-2 buffer and pSer2-Pol II antibody-conjugated beads were added. Incubated the IP reaction at 4 °C for 1 hr. The beads were washed with 1 mL of NET-2 buffer six times and 100 mL of 1xPNKT (1xPNK buffer and 0.05% Triton X-100) buffer once. Washed beads were incubated in 50 mL PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK (NEB)) in Thermomixer at 37 °C, 1,400 rpm for 6 min. Beads were washed with 1 mL of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was suspended in urea Dye (7M Urea, 1xTBE, 0.1% BPB and 0.1% XC) and resolved on 6% TBU gel (Invitrogen) at 200 V for 5 min. Gels with 30-160 nt RNAs were collected. 0.2 mL tube was prepared with holes made with 25G needle and placed in a 1.5 mL tube. Gel fragments were placed in the layered tube and broken down by centrifugation at 12,000 rpm for 1 min. The small RNAs were eluted from gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) at 25 °C for 1 hr in Thermomixer (900 rpm). Eluted RNA was purified with SpinX column (Coster) with 2 glass filters (Millipore) and the flow-through RNA was ethanol precipitated.
2019-10-15 | GSE134102 | GEO
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:Proteomic analysis of chick vestibualr hair bundles purified using the twist-off method. See Description.doc. Utricle hair bundles were purified from E20–21 chicks using the twist-off method (Gillespie & Hudspeth J Cell Biol 112, 625-640, 1991; Shin et al. Neuron 53, 371-386, 2007). NuPAGE LDS sample buffer (Invitrogen) with 50 mM dithiothreitol was added to a combined final volume of 80 µl per 100 utricles' worth of bundles; samples were heated to 70°C for 15 min. Epithelial proteins were similarly solubilized with sample buffer. Proteins were separated by running ~1 cm into a NuPAGE 4–12% Bis-Tris gel (1.5 mm x 10 well; one or two lanes per bundle sample); gels were rinsed with water, then stained with Imperial Protein Stain (Thermo Scientific). The 1 cm of gel with separated proteins was manually sliced into six pieces. Gel pieces were transferred to siliconized tubes and washed and destained in 200 µl 50% methanol overnight. The gel pieces were dehydrated in acetonitrile, rehydrated in 30 µl of 10 mM dithiothreitol in 0.1 M ammonium bicarbonate and reduced at room temperature for 0.5 h. The DTT solution was removed and the sample alkylated in 30 µl 50 mM iodoacetamide in 0.1 M ammonium bicarbonate at room temperature for 0.5 h. The reagent was removed and the gel pieces dehydrated in 100 µl acetonitrile. The acetonitrile was removed and the gel pieces rehydrated in 100 µl 0.1 M ammonium bicarbonate. The pieces were dehydrated in 100 µl acetonitrile, the acetonitrile removed and the pieces completely dried by vacuum centrifugation. The gel pieces were rehydrated in 20 ng/µl trypsin (Sigma-Aldrich T6567 proteomics grade, from porcine pancreas, dimethylated) in 50 mM ammonium bicarbonate on ice for 10 min. Any excess enzyme solution was removed and 20 µl 50 mM ammonium bicarbonate added. The sample was digested overnight at 37°C and the peptides formed extracted from the polyacrylamide in two 30 µl aliquots of 50% acetonitrile/5% formic acid. These extracts were combined and evaporated to 15 µl for MS analysis. For the gel slice immediately adjacent to the agarose, peptides were purified away from interfering polymers by SCX. A single experiment's worth of hair bundles or epithelium was analyzed by six LC-MS/MS runs, corresponding to the six gel pieces. The LC-MS/MS system consisted of a Thermo Electron Orbitrap Velos ETD mass spectrometer system with a Protana nanospray ion source, which was interfaced to a reversed-phase capillary column of 8 cm length x 75 µm internal diameter, self-packed with Phenomenex C18 Jupiter of 10 µm particle size. An extract aliquot (7.5 µl) was injected and peptides eluted from the column by an acetonitrile/0.1 M acetic acid gradient at a flow rate of 0.5 µl/min over 1.2 hr. The nanospray ion source was operated at 2.5 kV. The digest was analyzed using the data-dependent capability of the instrument, acquiring—in sequential scans—a single full scan mass spectrum in the Orbitrap detector at 60,000 resolution to determine peptide molecular weights, and 20 product-ion spectra in the ion trap to determine amino acid sequence. MaxQuant version 1.2.2.5 software was used for protein identification and quantitation. The default contaminants file associated with the MaxQuant download was edited to remove entries known to be present in hair bundles (e.g., actin) and to add additional impurities that entered the bundle-purification workflow (keratins, hemoglobins, egg white components). Mass spectrometry data were searched against Ensembl version 66 (released February, 2012) using Andromeda; the Ensembl FASTA file was edited by replacing several sequences with longer or full-length sequences, including actin gamma 1 (NP_001007825.1), actin beta (NP_990849.1), fascin 1 (NP_001171603), fascin 2 (NP_001171209), ATP synthase beta (NP_001026562.1), peptidylprolyl isomerase A (NP_001159798.1), calbindin 2 (NP_990647.1), PDZD7 (XP_003641537.1), espin (XP_417532.3), and CACNA2D2 (XP_427707.3). Protein identifications were reported with an FDR of 1%. If a set of peptides for a protein was identical to or completely contained within that of another protein, MaxQuant groups those proteins together ("redundant groups"); the entry with the largest number of peptides was used to identify the redundant group. Redundant groups that shared more than 20% of their identified peptides were further grouped in our analysis ("shared-peptide groups"); the entry with the greatest intensity associated with it was used to identify the shared-peptide group.
2013-01-08 | PXD000104 | Pride
Project description:Cells were treated with DMSO (biological triplicate) or degrader at indicated dose and time (Meta Treatment Table) and cells were harvested by centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, Protease and Phosphatase inhibitors from Roche) was added to the cell pellets and homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 1 - 4 mg/mL. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg of protein for each sample were reduced and alkylated as previously described1. Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme:protein) for 12 hours at room temperature. The LysC digestion was diluted to 0.5 M Urea with 200 mM EPPS pH 8 followed by digestion with trypsin (1:50; enzyme:protein) for 6 hours at 37 °C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 11 or 16-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. Each of the sample channels were combined in a 1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid and acidified (pH 2 - 3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions and these fractions were used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific) or an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an UltiMate 3000 RSLCnano System. Peptides were separated on an EasySpray ES803a/ES803a.rev2 75 μm inner diameter microcapillary column (Thermo Fisher Scientific) or a 100 µm inner diameter microcapillary column packed with ~ 50 cm of Accucore C18 resin (2.6 µM, 100 Å, Thermo Fisher Scientific). Peptides were separated using a 190 min gradient of 6 - 27% acetonitrile in 1.0% formic acid with a flow rate of 300 or 350 nL/min. Each analysis used an MS3-based TMT method as described previously. The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.
2021-07-30 | PXD023636 | Pride
Project description:Proteomic analysis Epilepsy was induced using perforant pathway stimulation (PPS) in rats as described (Norwood BA, et al. (2010) Classic hippocampal sclerosis and hippocampal-onset epilepsy produced by a single "cryptic" episode of focal hippocampal excitation in awake rats. J Comp 803 Neurol 518(16):3381-3407). Rats were killed under deep anesthesia (xylazine+ketamine) by transcardial perfusion with ice-cold 0.9 % NaCl solution at the following time points: 1 h, 24 h, 72 h, 10 d and 16 d after PPS (epileptogenesis); within 1 d of first spontaneous seizure (early epilepsy); 1 month after first spontaneous seizure (chronic epilepsy). Control rats were killed on day 17 after surgery (corresponding to day 10 after PPS). Hippocampi were removed and snap frozen at -80°C. Frozen rat hippocampi were resuspended in 200 ul lysis buffer (6M GdmCl, 10mM TCEP, Complete protease inhibitor cocktail, PhosSTOP inhibitor, 100 mM TEAB, benzonase) and dounced 30 times on ice. Samples were boiled for 5 min, sonicated on ice for 2min and centrifuged at 13000 rpm for 3 min. The supernatant was added chloroacetamide to a final concentration of 20 mM followed by incubation in the dark for 30 min at room temperature (RT). Proteins were digested with LysC for 30 min at RT and then diluting 10x— using 100 mM TEAB followed by adding trypsin and incubating overnight at 37°C with shaking. LysC and trypsin were added in a LysC/trypsin:protein ratio of 1:100. Samples were centrifuged at 13000 rpm for 5 min and from the supernatant a volume corresponding to 50 ug peptide was transferred to a new eppendorff tube. Sets of 6 samples were labelled using the TMTsixplex isobaric label reagent. (ThermoFisher Scientific). The labeling reagent was prepared using the manufactures instructions to first equilibrate to RT, then dissolving the labeling reagent in 41 ul anhydrous acetonitrile. The labeling reagent was added to each sample and incubated for 1 hour at RT. The reaction was quenched by incubating with 0.76 M lysine for 15 min. A small amount was taken from each sample, mixed and tested by mass spectrometry for labeling efficiency and mixing ratio. Remaining samples were mixed in a 1:1 ratio. The Stage-tips for the high-pH reverse phase fractionation were prepared by placing first two C18 disks in a p200 pipette tip, then adding a slurry of C18 beads (ReproSil-Pur, 3 um) in methanol and adding one additional C18 disk in the top. The column was activated and equilibrated by washing one time in 150 ul buffer B (50% buffer A, 50% ACN) and then two times in 150 ul buffer A (20mM Ammonium hydroxide, pH 10). The labeled peptide sample was adjusted to pH 10 using buffer A and then loaded onto the activated and equilibrated stage-tip by spinning the sample through the column and collecting 26 the flow-through. The column was washed 2 times with 150 ul buffer 585 A and the washes were collected and combined with the flow-through. Next, peptides were eluted stepwise in 17 fractions by spinning 150 ul of buffer A containing increasing amounts of ACN for each step (3.5%, 4.5%, 6%, 8%, 10%, 10.5%, 11.5%, 13.5%, 15%, 16%, 17.5%, 18.5%, 19.5%, 21%, 27%, 50%, 80%) through the column. After collection, the liquid was evaporated completely in a Concentrator Plus (Eppendorf) and prepared for MS by resuspending in buffer A* (2% ACN, 0.1% TFA). Samples were analyzed using an Easy-nLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source (Proxeon/Thermo Scientific). The peptides were eluted into the mass spectrometer during a chromatographic separation from a fused silica column packed in-house with 3 um C18 beads (Reprosil, Dr. Maisch) using a 120 min gradient of buffer B (80% ACN, 0.5% acetic acid) with a flow rate of 250 nL/min. The Q Exactive HF was operated in positive ion mode with a top 12 data-dependent acquisition method where ions were fragmented by higher-energy collisional dissociation (HCD) using a normalized collisional energy (NCE)/ stepped NCE of 28 and 32. The resolution was set to 60,000 (at 400m/z), with a scan range of 300-1700 m/z and an AGC target of 3e6 for the MS survey. MS/MS was performed at a scan range of 100 - 2000 m/z using a resolution of 30,000 (at 400 m/z), an AGC target of 1e5, an intensity threshold of 1e5 and an isolation window of 1.2 m/z. Further parameters included an exclusion time of 45 sec and a maximum injection time for survey and MS/MS of 15 ms and 45 ms respectively.
2020-06-12 | PXD019098 | Pride
Project description:This series represents the data set from the paper <u>Assembly of microarrays for genome-wide measurement of DNA copy number</u> appearing in Nat Genet 2001 Nov;29(3):263-4 and it's web supplement. Specimen procurement, labeling, hybridization and imaging protocols from Nat Genet 2001 Nov;29(3):263-4 web supplement: <b>Specimens.</b> We obtained GM03563, GM00143, GM05296, GM07408, GM01750, GM03134, GM13330, GM03576, GM01535, GM07081, GM02948, GM04435, GM10315, GM13031 and GM01524 cell strains from the NIGMS Human Genetics Cell Repository (Coriell Institute for Medical Research), and cultured them under the recommended conditions. We cultured cell lines BT474, HCT116, T47D, MPE600, COLO320, SW837, MDA-MB-231, MDA-MB-453 and HT29 under the recommended conditions. To isolate DNA from confluent cell cultures, we collected cells from T75 flasks and then incubated them overnight at 55 C in a 3 ml solution containing 0.01 M Tris, pH 7.5, 0.001 M EDTA, pH 8, 0.5% SDS and 0.1 g/l proteinase K. We added 1 ml of saturated NaCl solution and 10 ml of ethanol to the cell lysate and collected the DNA by spooling. We removed excess liquid and then dissolved the DNA in 1 ml of H2O. We isolated DNA from breast tumor specimens as described previously. <b>DNA labeling.</b> We labeled DNA by nick translation or random priming. For nick translation, we used a 400 l reaction containing 10 g DNA, 50 mM Tris, pH 7.6, 5 mM MgCl2, 0.02 mM each dATP, dTTP and dGTP, 0.2 mM Cy3- or Cy5-dCTP (Amersham), 0.2 U DNA Polymerase I (Gibco BRL), and 30 l DNAseI/PolI enzyme mix (Gibco BRL). We incubated the reaction at 15 C for 60 min after which we stopped the reaction by incubation at 70 C for 10 min. We removed unincorporated nucleotides using a Sephadex G-50 spin column. We labeled genomic DNA by random priming in a 100 l reaction containing 0.003 - 0.6 g DNA, 1x random primers solution (BioPrime DNA Labeling System, Gibco BRL), 1 mM Tris, pH 7.6, 0.1 mM EDTA, 0.2 mM each of dATP, dTTP and dGTP, 0.1 mM dCTP, 0.4 mM Cy3 or Cy5-dCTP (Amersham) and 160 U Klenow fragment (BioPrime DNA Labeling System, Gibco BRL). We incubated the DNA with the random primers solution at 100 C for 10 min in a total volume of 84 l, prior to adding the other reagents and then incubated the 100 l reaction overnight at 37 C. We removed unincorporated nucleotides using a Sephadex G-50 column. <b>Hybridization.</b> We combined test and reference DNAs (~2 g of input genomic DNA for nick translation or ~0.6 g of input genomic DNA for random prime labeling) with Cot-1 DNA (80-100 g; Gibco BRL) and precipitated them with ethanol. We collected the precipitate by centrifugation and allowed the precipitate to dry in air for 10 min before re-dissolving it in a 50 l hybridization mixture containing 50% formamide, 2 x SSC, 10% dextran sulfate, 4% SDS and 500 g yeast tRNA, pH 7. We incubated the hybridization mixture at 70 C for 10-15 min to denature the DNA and subsequently continued incubation at 37 C for 60 min to allow blocking of repetitive sequences. We applied a ring of rubber cement closely around the array to form a well, into which we added 50 l of slide blocking solution containing 500 g salmon sperm DNA in 50% formamide, 2 x SSC, 10% dextran sulfate and 4% SDS, pH 7. We created an airtight hybridization chamber by placing a silicone gasket (PGC Scientific) around the array and rubber cement ring, placing a slide on top of it and clamping with binder clips. After a 30 min incubation at room temperature, we opened the chamber, removed approximately three-quarters of the blocking solution, added the denatured and re-annealed hybridization mixture and then re-sealed the chamber. We placed the arrays on a slowly rocking table (~1 rpm) at 37 C to allow hybridization to occur over 16-72 h. After hybridization, we rinsed off the excess hybridization fluid with PN buffer (PN: 0.1 M sodium phosphate, 0.1% nonidet P40, pH 8), then washed once in 50% formamide, 2 x SSC, pH 7 at 45 C for 15 min, and finally in PN buffer at room temperature for 15 min. After draining excess liquid from the arrays, we mounted them in a solution containing 90% glycerol, 10% PBS and 1 M DAPI, and then sealed them with a cover slip. We drained excess glycerol-DAPI solution onto a paper tissue. <b>Imaging and analysis.</b> We acquired 16 bit 1024x1024 pixel DAPI, Cy3 and Cy5 images using a custom built CCD camera system, although we have also used commercial laser scanners for this purpose. We used ?UCSF SPOT? software (A.N.J. manuscript submitted) to automatically segment the spots based on the DAPI images, perform local background correction and to calculate various measurement parameters, including log2ratios of the total integrated Cy3 and Cy5 intensities for each spot. We used a second custom program SPROC to associate clone identities and a mapping information file with each spot so that the data could be plotted relative to the position of the BACs on the September, 2000 freeze of the draft human genome sequence (http://genome.ucsc.edu). SPROC also implements a filtering procedure to reject data based on a number of criteria, including low reference/DAPI signal intensity and low correlation of the Cy3 and Cy5 intensities with a spot. The SPROC output consists of averaged ratios of the triplicate spots for each clone, standard deviations of the triplicates and plotting position for each clone on the array, as well as other clone information stored in the database, such as STS content (Tables A and B). We edited the data files to remove ratios on clones for which only one of the triplicates remained after SPROC analysis and/or the standard deviation of the log2ratios of the triplicates was > 0.2. Keywords: other
2002-02-12 | GSE16 | GEO
Project description:2.3 Cell wall and secretome preparation. C. albicans RM1000 cells grown to mid-exponential phase were harvested by centrifugation. The resulting supernatant was sterile-filtered and then concentrated on 10-kDcut-off filters (Amicon Ultra-15 Centrifugal filter units, Millipore) as described previously [31]. To analyse the wall proteome, cell walls were isolated by hot SDS-extraction. The cell pellet obtained was used to prepare cell walls as described previously [36]. Briefly, the finely ground cell pellet was washed several times with PBS, and then subjected to breakage in a FastPrep bead beater (Savant Instruments Inc., Farmingdale, NY, USA) with glass beads (0.25-0.50 mm, 12-16 runs for 45 sec at speed 6) in the presence of a protease inhibitor cocktail. Full breakage was controlled by light-microscopic inspection. The pellet was washed several times with 1 M NaCl and stored overnight at 4°C. The following day, the pellet was washed several times with MilliQ-water and then boiled four times for 10 min in SDS extraction buffer (150 mM NaCl, 2% (w/v) SDS, 100 mM Na-EDTA, 100 mM β-mercaptoethanol, 50 mM Tris-HCl, pH 7.8), washed with MilliQ-water and lyophilized overnight. The resulting purified wall pellets were either stored at -80°C or directly reduced and S-alkylated [37]. 2.4 Mass spectrometric analyses of cell wall proteomes and secretomes. Lyophilized cell walls were reduced with 10 mM dithiothreitol in 100 mM NH4HCO3 (1 h at 55C). After cooling to room temperature and centrifugation, the supernatant was discarded. The reduced proteins were alkylated with 65 mM iodoacetamide in 100 mM NH4HCO3 for 45 min at room temperature in the dark. The samples were quenched with 55 mM dithiothreitol in 100 mM NH4HCO3 for 5 min. Subsequently, the samples were washed six times with 50 mM NH4HCO3 and either frozen in liquid nitrogen and stored at -80C or digested using 2 µg Trypsin Gold (Promega, Madison,WI) from a 1 µg/µl stock solution for 18 h at 37°C. The concentrated secretome samples were treated similarly, with reduction and alkylation on 10-kD cut-off spin filters (Amicon, Billerica, MA) [32]. The resulting tryptic digests were desalted using a C18 tip column (Varian, Palo Alto, CA) according to the manufacturer’s instructions. After evaporation of acetonitrile with a Speedvac (Genevac, Ipswich, England) the peptide concentration was determined at 205 nm using a NanoDrop ND-1000 (Isogen Life science, IJsselstein, The Netherlands) [38]. Each sample was diluted with 0.1% trifluoroacetic acid to a final concentration of 25 ng/µl, and 10 µl per run were injected onto an Ultimate 2000 nano-HPLC system (LC Packings, Amsterdam, The Netherlands) equipped with a PepMap100 C18 reversed phase column (75 µm inner diameter, 25 cm length; Dionex, Sunnyvale, CA). An elution flow rate of 0.3 µl/min was used along a linear gradient with increasing acetonitrile concentration over 45 min. The eluting peptides were directly ionized by electrospray in a Q-TOF (Micromass, Whyttenshawe, UK). Survey scans were acquired from m/z 350-1200. For low energy collision-induced dissociation (MS/MS), the most intense ions were selected in a data-dependent mode. 2.5 Data processing and analysis. After processing with the MaxEnt3 algorithm (MasslynxProteinlynx), the spectra were converted into pkl (peak list) files (available on PRIDE: www.ebi.ac.uk/pride: accession numbers 22716-22721) . Proteins were identified using MASCOT server 2.3.02 (Matrix Science, UK) and a database (6210 entries) consisting of a complete ORF translation of C. albicans and a list of regularly encountered contaminants. Enzyme was set to trypsin and allowing two miscleavages were allowed. Carbamidomethyl (C) was used as a fixed modification and Oxidation (M) as a variable modification. Mass tolerance and MS/MS tolerance were set to and a tolerance of 0.3 Da0.5 Da. Detected masses were charge deconvoluted to +1. Based on probabilistic MASCOT scoring a P value of < 0.05 was considered significant for peptide identification. At least three independently obtained biological samples were analysed for each condition and each was subjected to two MS/MS runs.
2013-02-20 | PXD000008 | Pride