PY phosphoproteomics in TNK1 cells Andersen
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Josh Anderson
PROVIDER: MSV000087623 | MassIVE | Tue Jun 15 05:59:00 BST 2021
REPOSITORIES: MassIVE
Similar Datasets
Project description:Qualitative LC MS/MS was performed on 4 uL of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 3 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed in a data dependent acquisition (DDA) mode of acquisition with a r=120,000 (@ m/z 200) full MS scan from m/z 375 to 1500 with a target AGC value of 2e5 ions. MS/MS scans were acquired at Rapid scan rate (Ion Trap) with an AGC target of 5e3 ions and a max injection time of 200 ms. The total cycle time for MS and MS/MS scans was 2 sec. A 20 sec dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours.
2021-06-14 | MSV000087618 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 2 uL of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed in a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 2e5 ions. MS/MS scans were acquired at Rapid scan rate (Ion Trap) with an AGC target of 5e3 ions and a max injection time of 100 ms. The total cycle time for MS and MS/MS scans was 2 sec. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours.
2022-12-05 | MSV000090835 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 2 uL using an MClass UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40v, -60v, -80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r 120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 1e4 and max fill time of 35 ms. The total cycle time for each CV was 0.66s, with total cycle times of 2 sec between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 hours.
2023-04-04 | MSV000091630 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 2 uL using an MClass UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40v, -60v, -80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r 120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 1e4 and max fill time of 35 ms. The total cycle time for each CV was 0.66s, with total cycle times of 2 sec between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 hou
2023-03-03 | MSV000091407 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 2 uL using an MClass UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40v, -60v, -80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r 120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 1e4 and max fill time of 35 ms. The total cycle time for each CV was 0.66s, with total cycle times of 2 sec between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 hours.
2023-03-09 | MSV000091457 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 2 uL of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipied with a FAIMS device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/ac etonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40, 60, 80). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (@ m/z 200) full MS scan from m/z 375-1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired at Rapid scan rate (Ion Trap) with an AGC target of 1e4 ions and a max injection time of 100 ms. The total cycle time for each CV was 0.66s, with total cycle times of 2 sec between like MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours.
2021-05-26 | MSV000087520 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 2 uL (1 ug) of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40v, -60v, -80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r 120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the Orbitrap at r 50,000 ( m/z 200) from m/z 100 with a target AGC value of 1e5 and max fill time of 35 ms. The total cycle time for each CV was 1s, with total cycle times of 3 sec between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours.
2023-11-27 | MSV000093511 | MassIVE
Project description:Quantitative LC-MS/MS was performed on 2.5 uL (6%) of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (40v, 60v, 80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the Orbitrap at r=50,000 (m/z 200) from m/z 100 with a target AGC value of 1e5 and max fill time of 35 ms. The total cycle time for each CV was 1s, with total cycle times of 3 sec between like full MS scans. A 45s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each fraction injection was approximately 2 hours.
Quantitative LC-MS/MS Analysis. Next, data was imported into Proteome Discoverer 2.5 (Thermo Scientific Inc.) and all LC-MS/MS runs were aligned based on the accurate mass and retention time of detected ions (features) which contained MS/MS spectra using Minora Feature Detector algorithm in Proteome Discoverer. Relative peptide abundance was calculated based on area under the curve (AUC) of the selected ion chromatograms of the aligned features across all runs.
2021-08-19 | MSV000088005 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 3 uL of each sample, using a nanoAcquity UPLC system (Waters Corp) coupled to a Thermo Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 ul/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed in a data-dependent acquisition (DDA) mode of acquisition with an r=120,000 (@ m/z 200) full MS scan from m/z 375 - 1500 and a target AGC value of 2e5 ions with a 2 sec cycle time. Ion trap MS/MS scans were acquired with a Rapid scan rate, 100 ms max injection time, and a target AGC value of 5e3 ions. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours.
Following 15 total UPLC-MS/MS analyses (excluding conditioning runs, but including 3 replicate QC injections; Table 1), data was imported into Proteome Discoverer 2.2 (Thermo Scientific Inc.), and analyses were aligned based on the accurate mass and retention time of detected ions (features) using Minora Feature Detector algorithm in Proteome Discoverer. Relative peptide abundance was calculated based on area-under-the-curve (AUC) of the selected ion chromatograms of the aligned features across all runs. The MS/MS data was searched against the TrEMBL D. rerio database (downloaded in May 2018) with additional proteins, including yeast ADH1, bovine serum albumin, as well as an equal number of reversed-sequence decoys) false discovery rate determination. Mascot Distiller and Mascot Server (v 2.5, Matrix Sciences) were utilized to produce fragment ion spectra and to perform the database searches. Database search parameters included fixed modification on Cys (carbamidomethyl) and variable modifications on Meth (oxidation) and Asn and Gln (deamidation). Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer were used to annotate the data at a maximum 1% protein false discovery rate.
2021-03-10 | MSV000087028 | MassIVE
Project description:Quantitative LC/MS/MS was performed on 2 uL of each fraction, using a nanoAcquity UPLC system (Waters Corp.) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) via a nanoelectrospray ionization source. Briefly, the fraction was first trapped on a Symmetry C18 20 mm x 180 um trapping column (5 uL/min at 99.9/0.1 v/v water/acetonitrile). Analytical separation was then performed using a 1.8 um Acquity HSS T3 C18 75 um x 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed in a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (m/z 200) full MS scan from m/z 375 - 1500 with a target AGC value of 2e5 ions.
MS/MS scans were acquired at Rapid scan rate (Ion Trap) with an AGC target of 5e3 ions and a max injection time of 100 milliseconds. The total cycle time for MS and MS/MS scans was 2 seconds. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each fraction injection was approximately 2 hours.
Following 35 total UPLC-MS/MS analyses (excluding conditioning runs, but including 3 replicate QC Pool (TotalPool), 4 replicate nuclear (NucPool) and 4 replicate cytosolic Pool (CytoPool) injections), data was imported into Proteome Discoverer 2.2 (Thermo Scientific Inc.). Analyses were aligned based on the accurate mass and retention time of detected ions (features) using Minora Feature Detector algorithm in Proteome Discoverer. Protein levels are based on the relative peptide abundance measures which were calculated by area-under-the-curve (AUC) of the selected ion chromatograms of the aligned features across all runs. Levels are reported in arbitrary units (a.u.)
Mascot Distiller and Mascot Server (v 2.5, Matrix Sciences) were utilized to produce fragment ion spectra from the MS/MS data and to perform database searches. The MS/MS data was searched against the SwissProt M. musculus database (downloaded in Apr 2017) to identify relevant M. musculus proteins. The MS/MS data was also searched against an equal number of reversed-sequence (decoys) for false discovery rate determination.. Database search parameters included fixed modification on Cys (carbamidomethyl) and , variable modifications on Meth (oxidation) and Asn and Gln (deamidation), full trypsin enzyme rules, and mass tolerances of 5ppm precursor ions and 0.8da product ions. Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer were used to annotate the data at a maximum 1% protein false discovery rate.
2021-07-07 | MSV000087774 | MassIVE