Targeting ERK-eIF4E Protein Translational Signaling in an Inflammatory Breast Cancer Model of Acquired Resistance and Reversal with an Altered Profile of Ribosomal Proteins Associated with Pathological Complete Response in Patients
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Homo Sapiens (ncbitaxon:9606)
SUBMITTER: 
Gayathri Devi
PROVIDER: MSV000094251 | MassIVE | Thu Mar 07 08:57:00 GMT 2024
SECONDARY ACCESSION(S): PXD050448
REPOSITORIES: MassIVE
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Project description:Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a Q-Exactive HF-X high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly the sample was first trapped on a Symmetry C18 180 micron x 20 mm trapping column (5 microliters/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 micron AQCUITY HSS T3 C18 75 micron x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on the HF-X MS was performed in data-dependent acquisition (DDA) mode with a 120,000 resolution (at m/z 200) full MS scan from m/z 375 to 1600 with a target AGC value of 3e6 ions and 50 ms maximum injection time (IT). The Top30 peptides were selected for MS/MS using a resolution of 15,000, max AGC of 5e4 ions and minimum AGC target of 2.25e3, max IT of 45 ms, collision energy of 27, an isolation width of 1.2 m/z and 20 s dynamic exclusion.
2019-11-07 | MSV000084557 | MassIVE
Project description:Heart tissue was lysed by sonication in 5% SDS (w/v) in 50 mM TEAB (pH 8.5) 450 microg of each sample was reduced with 10 mM DTT at 80 degC for 10 min and alkylated with 25 mM iodoacetamide in the dark for 30 min at room temperature. SDS was removed, and samples were digested with 20 microg of Sequencing Grade Modified Trypsin (Promega) using an S-trap mini device (Protifi) at 47 degC for 2 h. Lyophilized peptides were reconstituted in 100 microl of 200 mM TEAB buffer and labeled with 41 TMT10 reagents (lot# UL292365). After 2 h, reactions were quenched with hydroxylamine and combined.
TMT-labeled peptides were fractionated by high pH reversed-phase (HPRP) chromatography. Briefly, peptides were reconstituted in 400 microl of 20 mM ammonium formate, and 125 microl was fractionated using a 2.1 mm x 5 cm BEH C18 column (Waters) and Waters ACQUITY iClass UPLC. Separations utilized a flow rate of 0.4 ml/min and column temperature of 55 degC, and mobile phases consisted of 20 mM ammonium formate, pH 10 (MPA) and neat MeCN (MPB). Separations used a gradient as follows: 0 min, 3% B; 3 min, 7% B; 50 min, 50% B, 51 min, 90% B; 55 min, 90% B; 56 min, 3% B; 60 min, 3% B. Forty-eight equal fractions were collected over 52 minutes into a 96-well plate containing 10 microl of 20% TFA per well. This analysis was repeated 2 times total for fractionation of 2 mg peptides. The first 3 fractions of each injection were excluded, and the remaining samples were re-concatenated into 12 fractions. Approximately 5 microg of each fraction was separately aliquoted for analysis of the unenriched proteome, and samples were lyophilized.
Phosphopeptide enrichment used 200 microg of each of the 12 HPRP fractions was reconstituted in 80% MeCN/1% TFA containing 1M glycolic acid (buffer A), phosphopeptides were enriched using GL Sciences p10 TiO tips. After loading, tips were washed twice with buffer A followed by 2 times with 80% MeCN/1% TFA before elution with 20% MeCN/5% aqueous ammonia. After addition of neat formic acid, samples were lyophilized. Finally, peptides were desalted using C18 Stage Tips, lyophilized and reconstituted in 12 microl of 10 mM citrate in 1% TFA/2% MeCN. The unenriched fractions were reconstituted in 10 microl of 1% TFA/2% MeCN.
1D-LC-MS/MS was performed on 4.5 microl of each of the phospho-enriched fractions or on 0.75 microg of unenriched fractions. Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a coupled to a Exploris 480 high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source and FAIMS Pro Interface. Samples were first trapped on a Symmetry C18 180 microm x 20 mm trapping column (5 microl/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 microm Acquity HSS T3 C18 75 microm x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on was performed in data-dependent acquisition (DDA) mode with 3 FAIMS compensation voltages (-40, -60 and -80). Each CV used a 60,000 resolution full MS scan from m/z 375 to 1600 with a normalized AGC target of 300%, peptide monoisotopic peak determination, an intensity threshold of 5E3 ions, precursor fit of 70% with 0.7 m/z fit window, charge state of 2-5 and 40 s dynamic exclusion. MS/MS used a top6 method with 30,000 resolution and TurboTMT enabled, an isolation width of 0.7 m/z, NCE of 36, AGC target of 300% and maximum IT of 120 ms. Advanced precursor determination was enabled. The analysis of unenriched samples used a similar method except that a 1 s total scan time was used for each CV and MS/MS used an auto IT.
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Project description:Saliva was collected from n=3 subjects, before and after treatment with octreotide. 100 micrograms of saliva protein was reduced and alkylated, and digested with trypsin using an S-trap. Peptides (~45 ugs) were separated by microflow LC using a 1x100 mm Waters ACQUITY Premier CSH column at 100 uL/min using a gradient of 3-258 MeCN over 60 min. 3% DMSO was delivered post-column. The LC was interfaced to a Thermo Exploris 480 using a heated ESI source. MS/MS analysis used a staggered, overlapping DIA method with 60 K MS1 and 15K MS2 spanning 400-1000 m/z with 77 x 16 m/z windows and a 36 window cycle. Data was processed using Spectronaut Pulsar 15.
2022-03-13 | MSV000089053 | MassIVE
Project description:Cell lysates were adjusted to 5% SDS, and 20 ugs was reduced with 10 mM DTT and alkylated with 25 mM iodoacetamide followed by digestion with 1:15 trypsin using an S-trap at 47 degC for 1 h. Lyophilized peptides were reconstituted at 0.5 ugs/uL, and a study pool QC sample was made by mixing equal volumes of all samples. 1.75 uL of each sample was analyzed once, interspersed with 3 replicates of the SPQC pool, using a Waters M-Class in trap-elute configuration (75 uM x 25 cm HSS-T3 analytical column at 400 nl/min, 5-30% MeCN over 90 min) interfaced to a Thermo Exploris 480 with staggered overlapping window DIA MS analysis. Raw files were deconvoluted with .HTRMS converter (Biognosys) followed by direct-DIA and peptide/protein quantification with Spectronaut 16 (Biognosys).
2023-10-12 | MSV000093088 | MassIVE
Project description:As a pooled donor blood product, cryoprecipitate carries higher risks of pathogen transmission. Pathogen inactivation (PI) improves the safety of cryoprecipitate, but its effects on hemostatic properties remain unclear. This study investigated protein expression in samples of pathogen inactivated cryoprecipitate (PI-cryo) using non-targeted quantitative proteomics. Specifically, cryoprecipitate samples were reduced and alkylated and digested with trypsin. Approximately 50 micrograms of tryptic digests were analyzed by microflowLC-MS/MS. The LC separation used a Waters ACQUITY UPLC and a 1 mm x 150 mm 1.7 micron CSH C18 column using a flow rate of 100 microliters/min, a column temperature of 55C and a gradient of 3-28% (v/v) MeCN:H2O containing 0.1% formic acid over 60 minutes. The LC was interfaced to a Thermo Q-Exactive HF-X using the HESI-II source. The MS analysis used data-independent acquisition (DIA)-MS with a 120,000 resolution precursor ion (MS1) scan from 375-1500 m/z, AGC target of 3E6 and maximum injection time (IT) of 20 ms. DIA-MS/MS was performed using 30,000 resolution, AGC target of 3e6 and maximum IT of 60 ms, and 27 V NCE. The DIA windows included 15 x 37 m/z windows spanning 400-941 m/z with 1 m/z overlaps; as well as 3 x 87 m/z windows spanning 941-1200 m/z. Data was analyzed using Spectronaut Pulsar X
2020-05-04 | MSV000085374 | MassIVE
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:Fifteen microliters of plasma from sickle cell disease patients, and pooled samples, were diluted with 5% deoxycholate and 10 mM DTT, followed by heating at 80 degC for 30 min, alkylation with 25 mM iodoacteamide and digestion with modified trypsin for 4 h at 37 degC. After acidification, samples were filtered. ~35 micrograms of each sample was separated by direction injection and microflow LC (1 mm x 100 mm Waters ACQUITY Premier; 100 uL/min) using a gradient of 3-28% acetonrile, and MS/MS was acquired on a Thermo Exploris 480 using a 120K MS1 scan and 30K MS2 with a staggered/overlapping method. Data analysis used Spectronaut 17. Study pool QC data, and individual files used for statistical analysis, are described in the accompanying metadata.
2024-01-19 | MSV000093893 | MassIVE
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: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:HT22 cells were cultured under hypoxia for 17.5 h with 0.69 mM low glucose (H+LG), hypoxia without glucose, or normoxia with normal 22 mM glucose (N+G). Proteomic analysis was performed by the Duke Proteomics and Metabolomics Shared Resource (address correspondence to mwfoster(at)duke.edu. Cells (n=3 replicates per condition) were lysed by sonication in 0.5% ALS-1, reduced with DTT and alkylated with iodoacetamide followed by digestion with trypsin digestion overnight. A QC pool was made by mixing equal amounts of all samples. Samples were analyzed using a nanoACQUITY UPLC system (Waters) coupled to a Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. 750 ng of each sample was analyzed using block randomization with interspersed analyses of the QC pool. Briefly, the sample was first trapped on a Symmetry C18 180 micron by 20 mm trapping column (5 microl/min at 99.9/0.1 v/v H2O/MeCN) followed by an analytical separation using a 1.7 micron AQCUITY HSS T3 C18 75 micron x 250 mm column (Waters) with a 90 min gradient of 5 to 30% MeCN with 0.1% formic acid at a flow rate of 400 nl/min and column temperature of 55 degC. Data collection on the Fusion Lumos MS was performed in data-dependent acquisition (DDA) mode with a 240,000 resolution (at m/z 200) full MS scan from m/z 375 to 1600 with a target AGC value of 2e5 ions and 50 ms maximum injection time (IT) with internal calibration enabled. Peptides were selected for MS/MS using with advanced peak determination enabled, peptide monosotopic peak determination, and including charge states 2-5. MS/MS used HCD fragmentation and detection in the ion trap. Briefly, a 2 s method used an isolation width of 0.7 m/z, a normalized collision energy of 30 plus/minus 5%, a rapid ion trap scan rate, max AGC of 5e3 ions, max IT of 300 ms and use of all available parallelization time. A 20 s dynamic exclusion was enabled. Data was analyzed using Rosetta Elucidator v.4 (see Supplemental Data). Database searching with Mascot Server v 2.5 used a Swissprot Mus Musculus Database (downloaded 09/05/2017) appended with common contaminants and reverse decoys. Search parameters included precursor mass tolerance of 5 ppm, product ion mass tolerance of 0.6 Da, trypsin
specificity with up to two missed cleavages, fixed modification on Cys (carbamidomethyl) and variable deamidation (N/Q)and acetylation (protein N-terminus). Data was annotated at a 1% peptide FDR using the PeptideTeller algorithm in Elucidator. Peptides were quantified by area under the curve and normalized to robust mean, and protein intensities were calculated as the sum of peptide intensities.
2020-08-15 | MSV000085952 | MassIVE