Data

Proteomics data for androgen receptor splice variant-7 in circulating tumour cell project, led by Therese Becker

Western Sydney University
Becker, Therese ; Harman, David ; Scott, Kieran ; Khan, Tanzila ; Lock, John ; Ma, Yafeng ; de Souza, Paul ; Chua, Wei ; Balakrishnar, Bavanthi
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ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rfr_id=info%3Asid%2FANDS&rft_id=info:doi10.26183/7d43-ze13&rft.title=Proteomics data for androgen receptor splice variant-7 in circulating tumour cell project, led by Therese Becker&rft.identifier=10.26183/7d43-ze13&rft.publisher=Western Sydney University&rft.description=The dataset contains the raw data for samples 7 and 8, proteins digested from a gel band excised from proteins extracted from PC3 cells. It also contains the ProteinLynx Global Server (Waters) search results of the proteins identified. Total protein lysate from PC3 prostate cancer cells was separated on 4-12% SDS page gels and a band corresponding to approximately 62kDa was excised and Mass Spectroscopy performed to test for any peptide homology to the androgen receptor (AR) or its variant 7 (AR-V7). No homology was detected. Mass spectrometric sample preparation was as follows: Gel spots were placed in 500 µL protein Lo-Bind tubes (Eppendorf). Proteins were fixed by treatment with 200 µL of 10% MeOH and 7% acetic acid in ultrapure water (20 mins), then spots werre washed twice with 200 µL of water. Dehydration was performed by speedivac and the shrunken spots treated with 100 µL of 10 mM DTT in 100 mM aqueous ammonium bicarbonate. After heating at 60°C for 45 min, supernatants were aspirated and discarded. Alkylation was accomplished by the addition of 100 µL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate, for 30 mins at room temperature. Gel pieces were then washed with 100 µL of 100 mM ammonium bicarbonate and dehydrated once more via speedivac. Dried spots were treated with 20 µL of trypsin (10 ng/µL) in 100 mM ammonium bicarbonate and place on ice for 45 min while rehydrating. Digestion was continued at 22°C for 17 hr. Supernatants were aspirated and placed in fresh 500 µL tubes. Remaining peptides were extracted twice with 50 µL of 50% aqueous acetonitrile with 2% formic acid, with bath sonication for 10 mins. Combined extracts were evaporated by speedivac and peptides were reconstituted with 15 µL of 0.1% aqueous formic acid, using sonication (10 min). Tubes were centrifuged for 10 min at 14k rpm and the supernatants placed in Total Recovery chromatography vials (Thermo). The digested peptides were separated by UPLC-MS using a Waters nanoAcquity UPLC sample manager fitted with a binary solvent manager. Separation consisted of two mobile phases. Mobile Phase A (0.1% formic acid in Milli-Q water) and Mobile Phase B (0.1% formic acid in ACN). The trapping column was a Waters nanoEase M/Z Symmetry C18 trap column (180 µm x 20 mm) and the analytical column was a Waters nanoAcquity UPLC 1.7um BEH130 C18 column (75 µm x 100 mm) thermostatted to 35 °C. Elution was achieved at a flow rate of 300 nL/min with each sample run for 50 minutes. The gradient was 0 min 1% B; 2 min 10% B; 40 min 40% B; 42 min 85% B and 50 min 85% B. Samples were suspended in 0.1% formic acid and 1 µL injected for each run. Mass spectrometric detection was conducted using a Waters Synapt G2-Si, fitted with a nanoESI source, run in positive ion mode with a capillary voltage of 3 kV and a sampling cone voltage of 30 V as well as a source offset of 30 V for electrospray ionizations. The source temperature was set at 80 °C. A desolvation flow of nitrogen gas at 600 l/h and a desolvation temperature of 350 °C was used. Lock spray configuration was conducted every 60 seconds with [Glu1]-fibrinopeptide B as the reference compound. Data acquisition was conducted over the mass to charge range of 50–2000. The data independent acquisition used an MSE experiment employing both low and high energy collision-induced dissociation of parent ions. Low energy collision was done at 6 V in the trap collision cell and at 4 V in the transfer collision cell. High energy collision used a collision energy ramp from 17 V to 60 V in the transfer collision cell. Scan time was 0.5 seconds and after each scan the system would switch from high to low energy collision. Analysis of raw data was conducted using ProteinLynx Global Server (Waters), searching against the UniProt database using a Human taxonomic restriction. Variable modifications of carbamidomethyl (C), deamidated (NQ), oxidation (M) and propionamide (C) were used.&rft.creator=Becker, Therese &rft.creator=Harman, David &rft.creator=Scott, Kieran &rft.creator=Khan, Tanzila &rft.creator=Lock, John &rft.creator=Ma, Yafeng &rft.creator=de Souza, Paul &rft.creator=Chua, Wei &rft.creator=Balakrishnar, Bavanthi &rft.date=2022&rft.edition=undefined&rft_rights=Copyright Western Sydney University&rft_rights=CC BY 4.0: Attribution 4.0 International http://creativecommons.org/licenses/by/4.0&rft_subject=androgen receptor deprivation therapy&rft_subject=androgen receptor splice variant-7&rft_subject=antibody specificity, biomarker&rft_subject=castrate resistant prostate cancer&rft_subject=circulating tumor cells&rft_subject=liquid biopsy&rft.type=dataset&rft.language=English ctx_ver=Z39.88-2004&rft_val_fmt=info%3Aofi%2Ffmt%3Akev%3Amtx%3Adc&rfr_id=info%3Asid%2FANDS&rft_id=info:doi10.26183/7d43-ze13&rft.title=Proteomics data for androgen receptor splice variant-7 in circulating tumour cell project, led by Therese Becker&rft.identifier=10.26183/7d43-ze13&rft.publisher=Western Sydney University&rft.description=The dataset contains the raw data for samples 7 and 8, proteins digested from a gel band excised from proteins extracted from PC3 cells. It also contains the ProteinLynx Global Server (Waters) search results of the proteins identified. Total protein lysate from PC3 prostate cancer cells was separated on 4-12% SDS page gels and a band corresponding to approximately 62kDa was excised and Mass Spectroscopy performed to test for any peptide homology to the androgen receptor (AR) or its variant 7 (AR-V7). No homology was detected. Mass spectrometric sample preparation was as follows: Gel spots were placed in 500 µL protein Lo-Bind tubes (Eppendorf). Proteins were fixed by treatment with 200 µL of 10% MeOH and 7% acetic acid in ultrapure water (20 mins), then spots werre washed twice with 200 µL of water. Dehydration was performed by speedivac and the shrunken spots treated with 100 µL of 10 mM DTT in 100 mM aqueous ammonium bicarbonate. After heating at 60°C for 45 min, supernatants were aspirated and discarded. Alkylation was accomplished by the addition of 100 µL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate, for 30 mins at room temperature. Gel pieces were then washed with 100 µL of 100 mM ammonium bicarbonate and dehydrated once more via speedivac. Dried spots were treated with 20 µL of trypsin (10 ng/µL) in 100 mM ammonium bicarbonate and place on ice for 45 min while rehydrating. Digestion was continued at 22°C for 17 hr. Supernatants were aspirated and placed in fresh 500 µL tubes. Remaining peptides were extracted twice with 50 µL of 50% aqueous acetonitrile with 2% formic acid, with bath sonication for 10 mins. Combined extracts were evaporated by speedivac and peptides were reconstituted with 15 µL of 0.1% aqueous formic acid, using sonication (10 min). Tubes were centrifuged for 10 min at 14k rpm and the supernatants placed in Total Recovery chromatography vials (Thermo). The digested peptides were separated by UPLC-MS using a Waters nanoAcquity UPLC sample manager fitted with a binary solvent manager. Separation consisted of two mobile phases. Mobile Phase A (0.1% formic acid in Milli-Q water) and Mobile Phase B (0.1% formic acid in ACN). The trapping column was a Waters nanoEase M/Z Symmetry C18 trap column (180 µm x 20 mm) and the analytical column was a Waters nanoAcquity UPLC 1.7um BEH130 C18 column (75 µm x 100 mm) thermostatted to 35 °C. Elution was achieved at a flow rate of 300 nL/min with each sample run for 50 minutes. The gradient was 0 min 1% B; 2 min 10% B; 40 min 40% B; 42 min 85% B and 50 min 85% B. Samples were suspended in 0.1% formic acid and 1 µL injected for each run. Mass spectrometric detection was conducted using a Waters Synapt G2-Si, fitted with a nanoESI source, run in positive ion mode with a capillary voltage of 3 kV and a sampling cone voltage of 30 V as well as a source offset of 30 V for electrospray ionizations. The source temperature was set at 80 °C. A desolvation flow of nitrogen gas at 600 l/h and a desolvation temperature of 350 °C was used. Lock spray configuration was conducted every 60 seconds with [Glu1]-fibrinopeptide B as the reference compound. Data acquisition was conducted over the mass to charge range of 50–2000. The data independent acquisition used an MSE experiment employing both low and high energy collision-induced dissociation of parent ions. Low energy collision was done at 6 V in the trap collision cell and at 4 V in the transfer collision cell. High energy collision used a collision energy ramp from 17 V to 60 V in the transfer collision cell. Scan time was 0.5 seconds and after each scan the system would switch from high to low energy collision. Analysis of raw data was conducted using ProteinLynx Global Server (Waters), searching against the UniProt database using a Human taxonomic restriction. Variable modifications of carbamidomethyl (C), deamidated (NQ), oxidation (M) and propionamide (C) were used.&rft.creator=Becker, Therese &rft.creator=Harman, David &rft.creator=Scott, Kieran &rft.creator=Khan, Tanzila &rft.creator=Lock, John &rft.creator=Ma, Yafeng &rft.creator=de Souza, Paul &rft.creator=Chua, Wei &rft.creator=Balakrishnar, Bavanthi &rft.date=2022&rft.edition=undefined&rft_rights=Copyright Western Sydney University&rft_rights=CC BY 4.0: Attribution 4.0 International http://creativecommons.org/licenses/by/4.0&rft_subject=androgen receptor deprivation therapy&rft_subject=androgen receptor splice variant-7&rft_subject=antibody specificity, biomarker&rft_subject=castrate resistant prostate cancer&rft_subject=circulating tumor cells&rft_subject=liquid biopsy&rft.type=dataset&rft.language=English Access the data

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The dataset contains the raw data for samples 7 and 8, proteins digested from a gel band excised from proteins extracted from PC3 cells. It also contains the ProteinLynx Global Server (Waters) search results of the proteins identified. Total protein lysate from PC3 prostate cancer cells was separated on 4-12% SDS page gels and a band corresponding to approximately 62kDa was excised and Mass Spectroscopy performed to test for any peptide homology to the androgen receptor (AR) or its variant 7 (AR-V7). No homology was detected. Mass spectrometric sample preparation was as follows: Gel spots were placed in 500 µL protein Lo-Bind tubes (Eppendorf). Proteins were fixed by treatment with 200 µL of 10% MeOH and 7% acetic acid in ultrapure water (20 mins), then spots werre washed twice with 200 µL of water. Dehydration was performed by speedivac and the shrunken spots treated with 100 µL of 10 mM DTT in 100 mM aqueous ammonium bicarbonate. After heating at 60°C for 45 min, supernatants were aspirated and discarded. Alkylation was accomplished by the addition of 100 µL of 50 mM iodoacetamide in 100 mM ammonium bicarbonate, for 30 mins at room temperature. Gel pieces were then washed with 100 µL of 100 mM ammonium bicarbonate and dehydrated once more via speedivac. Dried spots were treated with 20 µL of trypsin (10 ng/µL) in 100 mM ammonium bicarbonate and place on ice for 45 min while rehydrating. Digestion was continued at 22°C for 17 hr. Supernatants were aspirated and placed in fresh 500 µL tubes. Remaining peptides were extracted twice with 50 µL of 50% aqueous acetonitrile with 2% formic acid, with bath sonication for 10 mins. Combined extracts were evaporated by speedivac and peptides were reconstituted with 15 µL of 0.1% aqueous formic acid, using sonication (10 min). Tubes were centrifuged for 10 min at 14k rpm and the supernatants placed in Total Recovery chromatography vials (Thermo). The digested peptides were separated by UPLC-MS using a Waters nanoAcquity UPLC sample manager fitted with a binary solvent manager. Separation consisted of two mobile phases. Mobile Phase A (0.1% formic acid in Milli-Q water) and Mobile Phase B (0.1% formic acid in ACN). The trapping column was a Waters nanoEase M/Z Symmetry C18 trap column (180 µm x 20 mm) and the analytical column was a Waters nanoAcquity UPLC 1.7um BEH130 C18 column (75 µm x 100 mm) thermostatted to 35 °C. Elution was achieved at a flow rate of 300 nL/min with each sample run for 50 minutes. The gradient was 0 min 1% B; 2 min 10% B; 40 min 40% B; 42 min 85% B and 50 min 85% B. Samples were suspended in 0.1% formic acid and 1 µL injected for each run. Mass spectrometric detection was conducted using a Waters Synapt G2-Si, fitted with a nanoESI source, run in positive ion mode with a capillary voltage of 3 kV and a sampling cone voltage of 30 V as well as a source offset of 30 V for electrospray ionizations. The source temperature was set at 80 °C. A desolvation flow of nitrogen gas at 600 l/h and a desolvation temperature of 350 °C was used. Lock spray configuration was conducted every 60 seconds with [Glu1]-fibrinopeptide B as the reference compound. Data acquisition was conducted over the mass to charge range of 50–2000. The data independent acquisition used an MSE experiment employing both low and high energy collision-induced dissociation of parent ions. Low energy collision was done at 6 V in the trap collision cell and at 4 V in the transfer collision cell. High energy collision used a collision energy ramp from 17 V to 60 V in the transfer collision cell. Scan time was 0.5 seconds and after each scan the system would switch from high to low energy collision. Analysis of raw data was conducted using ProteinLynx Global Server (Waters), searching against the UniProt database using a Human taxonomic restriction. Variable modifications of carbamidomethyl (C), deamidated (NQ), oxidation (M) and propionamide (C) were used."

Created: 2022-05-26

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  • Local : https://research-data.westernsydney.edu.au/published/cc59f230dcb911ecb51f39ebd3ab2f2a
  • DOI : 10.26183/7d43-ze13