Presynaptic Rac1 Interactome in vivo BioID Soderling and Kim
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Astral
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Scott Soderling
PROVIDER: MSV000095110 | MassIVE | Fri Jun 21 12:18:00 BST 2024
REPOSITORIES: MassIVE
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Project description:Ten micrograms of each sample were reduced and alkylated followed by trypsin digestion using a suspension trap (S-trap). An SPQC was made by combining equal volumes of each sample. After lyophilization and reconstitution of the sample in 20 uL, 1-2 uL of each was analyzed by LC-MS/MS using a 30 min gradient on a Thermo Vanquish Neo coupled to a Thermo Orbitrap Astral via a Nanospray Flex ionization source.
Briefly, the sample was first trapped on a PepMap C18 0.3 mm x 5 mm trapping column (50 ul/min at 99.9/0.1 v/v water/acetonitrile), followed by analytical separation using a 1.5 um 150um ID x 8cm (PepSep) column with a 20 min gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 500 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Orbitrap Astral mass spectrometer was performed in a data-independent acquisition (DIA) mode of acquisition with a r=240,000 (@ m/z 200) full MS scan every 0.6s from m/z 380-980 with an AGC target of 500%. DIA MS/MS scans were acquired in the Astral analyzer using fixed windows of 4 m/z from m/z 380, target AGC of 500% and max fill time of 6 ms. HCD collision energy setting of 27% was used for all MS2 scans.
Raw MS data was demultiplexed and converted to *.htrms format using HTRMS converter and processed in Spectronaut 18 (Biognosys). A spectral library was built using direct-DIA searches which used a Mus muculus database, downloaded from Uniprot on 4/08/23, and appended the transgene APOBEC1-YTH as well as contaminant sequences using FragPipe. Search settings included N-terminal trypsin/P specificity up to 2 missed cleavages; peptide length from 7-52 amino acids with the following modifications: acetyl (N-term), carbamidomethyl(Cys) and oxidation (M). For DIA analysis, default extraction, calibration, identification and protein inference settings were used. Normalization, imputation and protein quantification was performed using an in-house script.
2024-03-21 | MSV000094375 | MassIVE
Project description:We highlight the leap in performance made possible with new instrumentation in the Thermo Scientific Orbitrap Astral™ mass spectrometer. Integrated alongside a high-resolution accurate mass (HRAM) Orbitrap analyzer, the instrument introduces a fast-switching quadrupole mass filter congruent with downstream speeds of ion detection, a dual-pressure ion processor cell which fragments ions in the high-pressure cell before they are moved to the low-pressure cell for further accumulation and orthogonal extraction to the Astral analyzer, and a high dynamic range detector. Altogether, ions can be manipulated in five stages of the MS at once (Orbitrap, ion routing multipole, two ion processor stages, Astral analyzer), allowing for a high degree of parallelization which enables MS/MS scan speeds of 200 Hz while HRAM MS1 full scans are synchronously acquired in the Orbitrap analyzer. To provide such fast scan speeds, the Astral analyzer is capable of single-ion detection sensitivity like a linear ion trap, all while enabling resolving powers of 80,000 at m/z 524. Here, in triplicate 7-min microflow active LC gradients on the Orbitrap Astral MS, we report 7,852 protein groups from 94,267 peptides on average. When employing 15-, 30-, and 60-min active, nano-LC gradients, triplicate experiments yield an average of 9,831, 10,411, and 10,645 unique protein groups from 195,612, 234,406, and 245,754 unique peptides, respectively. These nanoflow methods delivered a median sequence coverage of 38%, 42.4% and 44.4% across identified proteins for each respective gradient length. Our 30-min method delivered approximately 347 proteins per minute. Finally, we applied our 30-min active gradient method to analyze a CRISPR-Cas9 knockout (KO) of the mitochondrial gene MGME1 in human HAP1 cells. There, we quantified an average of 10,353 proteins across three technical replicates and find 449 significantly up- or down-regulated proteins relative to wild-type (WT) HAP1 proteins. Proteins from the same sample quantified using a 61-min active gradient Orbitrap Ascend DIA method show strong fold-change correlations to and gene ontology (GO) term agreement with our results.Building upon the immense progress of the mass spectrometry and proteomics communities of the past ten years, here we report alongside other recent works, that the one-hour human proteome is now within reach.
2024-03-29 | PXD049028 | Pride
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 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 - 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.
2021-06-15 | MSV000087623 | MassIVE
Project description:Quantitative 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) 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 linear 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 sample injection was approximately 2 hours.
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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.
2023-07-24 | MSV000092512 | 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.
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