Quantitative proteomics of ECM1-knockdown mouse kidney after chronic kidney disease
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Eclipse
ORGANISM(S): Mus Musculus (ncbitaxon:10090)
SUBMITTER: 
Dong Zhou
PROVIDER: MSV000090191 | MassIVE | Tue Aug 23 12:29:00 BST 2022
REPOSITORIES: MassIVE
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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:Serum samples were inactivated and sterilized at 56 ℃ for 30 min and processed as previous study with some modifications (Shen et al., 2020). Briefly, 4 μL serum from each sample was depleted using High Select Top-14 Abundant Protein Depletion MiniSpin Columns (Thermo Fisher Scientific, San Jose, USA). Then the elutes were denatured, reduced and alkylated to derive protein lysates. The solutions were diluted with 200 μL 100 mM triethylammonium bicarbonate (TEAB) followed by a double-step trypsinization (enzyme-to-substrate ratio kept at 1:40 in each step). 10% trifluoroacetic acid (TFA) was added to each sample to quench the enzymatic reaction. Digested peptides were cleaned-up with SOLAμ (Thermo Fisher Scientific, San Jose, USA) according to the manufacturer’s instructions and labeled by TMTpro 16 plex reagents (Thermo Fisher Scientific, San Jose, USA). 660 samples in total (633 serum samples and 27 technical replicates) were divided into 44 batches for labelling experiment, each batch containing fifteen samples and one pool. The labeled samples in each batch were combined and fractionated. Each sample was separated into 30 fractions and then consolidated into 10 fractions. Dried and re-dissolved fractions with 2% acetonitrile (ACN)/0.1% formic acid (FA) of MS grade. The re-dissolved peptides were analyzed with a U3000 HPLC system coupled to a Orbitrap Exploris 480 (Thermo Fisher Scientific, Bremen, Germany) in data-dependent acquisition (DDA) mode combined to a front-end high-field asymmetric waveform ion mobility spectrometry (FAIMS). Peptides of each fraction were loaded onto a pre-column (3 μm, 100 Å, 20 mm*75 mm i.d.) and separated with a 75 min LC gradient at a flow rate of 300 nL/min (analytical column: 1.9 mm, 120 Å, 150 mm*75 mm i.d.; Buffer A: 2% ACN, 98% H2O containing 0.1% FA; Buffer B: 98% ACN, 2% H2O containing 0.1% FA). FAIMS was operated at two different CV, -48 V and -68 V, respectively. For MS acquisition, RF level was set at 50% and ion transfer tube temperature at 320 °C. The turbo-TMT mode was enabled. Scan range of MS1 was 350–1800 m/z. Resolutions were set to 60000 for MS1 and 30000 for MS2. Normalized AGC target was set at 300% for MS1 and 200% for MS2. The maximum injection time was set as 50 ms for MS1 and 86 ms for MS2. Dynamic exclusion was on and mass tolerance was set to ±10 ppm. Intensity threshold was set at 20000 for MS1, and HCD collision energy was fixed to 38%. MS/MS data was recorded in centroid mode. Isolation window was set to 0.7 m/z.
2022-10-06 | PXD037204 |
Project description:The cell lysates were mixed with anti-FLAG-antibody-crosslinked Dynabeads. The beads-lysate mix was incubated at 4°C for 45 min. After washing with PBS-T, proteins were eluted with Laemmli’s SDS ample buffer by incubating at 37°C for 30 min. The collected supernatants were frozen at -80°C as samples for LC-MS. After reduction with 10 mM TCEP at 100°C for 10 min and alkylation with 50 mM iodoacetamide at ambient temperature for 45 min, the protein samples were subjected to digestion with Trypsin/Lys-C Mix (Promega) at 47°C for 2 h on S-Trap columns (ProtiFi, NY, USA).
The resulting peptides were extracted from gel fragments and analysed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) combined with UltiMate 3000 RSLC nano-flow HPLC (Thermo Scientific). The peptides were enriched with μ-Precolumn (0.3 mm i.d. × 5 mm, 5 μm, Thermo Scientific) and separated on an AURORA column (0.075 mm i.d. × 250 mm, 1.6 μm, Ion Opticks Pty Ltd, Australia) using the two-step gradient 2-40% acetonitrile for 110 min, followed by 40-95% acetonitrile for 5 min in the presence of 0.1% formic acid. The compensation voltages for the gas-phase fractionation by FAIMS Pro (Thermo Scientific) were set at -40, -60 and -80 V. The analytical parameters of the Orbitrap Fusion Lumos were set as follows: resolution of full scans = 50,000, scan range (m/z) = 350-1500, maximum injection time of full scans = 50 ms, AGC target of full scans = 4 × 10^5, dynamic exclusion duration = 30 s, cycle time of data dependent MS/MS acquisition = 2 s, activation type = HCD, detector of MS/MS = ion trap, maximum injection time of MS/MS = 35 ms, and AGC target of MS/MS = 1 × 10^4.
The MS/MS spectra were searched against the Homo sapiens protein sequence database in SwissProt using Proteome Discoverer 3.0 software (Thermo Scientific), in which peptide identification filters were set at “false discovery rate < 1%”. Label-free relative quantification analysis for proteins was performed with the default parameters of Minora Feature Detector node, Feature Mapper node, and Precursor Ions Quantifier node in Proteome Discoverer 3.0 software.
2024-10-30 | PXD056735 | JPOST Repository
Project description:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.
2021-07-12 | PXD025754 | Pride
Project description:The cells were grown on Fe(II)-citrate as the aqueous Fe(II) source and Fe(II)-smectite as the solid Fe(II) source. Cells were collected on a 0.2 um filter membrane (Millipore GPWP) by filtration. Then cells on the membrane were lysed by vortex in the lysis buffer containing 100 mM Tris/HCl, 10 mM Ethylenediaminetetraacetic acid (EDTA), pH 8.0, 0.05% Tween-20 and 1% w/v sodium dodecylsulfate (SDS). The proteins were digested with trypsin using in-house packed glass fiber filters following the suspension trapping (STrap) protocol, and desalted using C18-based StageTips. The LC-MS/MS analysis was performed using an Orbitrap Eclipse MS (Thermo Scientific) coupled with an Ultimate 3000 nanoLC system and a FAIMS Pro Interface (Thermo Scientific). Peptides were first loaded onto a trap column (PepMap C18; 2 cm x 100 um I.D.) and then separated by an analytical column (PepMap C18, 3.0 um; 10 cm x 75 um I.D.; Thermo Scientific) at 300 nl/min flow rate using a binary buffer system (buffer A, 0.1% formic acid in water; buffer B, 0.1% formic acid in acetonitrile) with a 165-min gradient (1% to 10% in 8 min; then to 25% buffer B over 117 min; 25% to 32% buffer B in 10 min, then to 95% buffer B over 3 min; back to 1% B in 5 min, and stay equilibration at 1% B for 20 min). Multiple CVs (-50, -65 and -80) were applied for FAIMS separation. For all experiments, the survey scans (MS1) were acquired over a mass range of 375-1500 m/z at a resolution of 60,000 in the Orbitrap. The maximum injection time was set to Auto, and AGC target was set to Standard. Monoisotopic peak selection was set to Peptides, and the charge state filter was set to 2-7. For MS/MS acquisition, precursors were isolated with a width of 1.6 m/z, fragmented with HCD using 30% collision energy with a maximum injection time of 100 ms, and collected in Orbitrap at 15,000 resolution. The dynamic exclusion was set to 60 s, and can be shared across different FAIMS experiments. Protein identification and quantitation were performed using the MaxQuant-Andromeda software suite (version 1.6.3.4) with most of the default parameters. An UniProt database (Sideroxydans lithotrophicus ES-1; Taxonomy ID, 580332; 2,978 sequences) was used for the protein identification. Other parameters include: trypsin as an enzyme with maximally two missed cleavage sites; protein N-terminal acetylation and methionine oxidation as variable modifications; cysteine carbamidomethylation as a fixed modification; peptide length must be at least 7 amino acids. False discovery rate was set at 1% for both proteins and peptides. Perseus (version 1.6.7.8) was used for downstream bioinformatics analysis such as clustering, correlation, t-test and ANOVA.
2022-03-29 | MSV000089149 | MassIVE
Project description:Proteome quantification using TMT reagents and off-line basic peptide fractionation was done as described previously in Grand, 2021, with the exception that the isolated nuclei were used as a starting material rather than whole cells to enable detection of transcription factors which are generally less abundant proteins in the cell, and that nine channels from a TMTpro16 reagent kit were used. Briefly, mESCs, NGN2 24h induced cells and MyoD1 24h induced cells were harvested in triplicates of 10 million per sample. Nuclei were isolated by suspending in 4 ml of nuclear extraction buffer (10 mM HEPES pH 7.9, 10 mM KCl, 10 mM EDTA, 0.5% NP-40, 1 mM DTT and complete protease inhibitor (Roche)) and incubating on ice for 10 min with vortexing every 2 minutes. Nuclei were collected by centrifugation (800g, 10 min, 4 °C), washed with ice cold PBS and suspended in 1% sodium deoxycholate in 50 mM HEPES buffer (pH 8.5). The nuclear lysate was disrupted with sonication. Proteins were reduced, alkylated and Lys-C/trypsin digested. Fifty micrograms of peptides from each biological replicate were labelled using channels 128C-131C from a TMT 16plex isobaric labelling reagents kit (Lot# VJ313476, Thermo Fisher Scientific). Dried TMT-labeled peptides were dissolved in 20 μL of 10 mM ammonium formate (pH 10, high pH Buffer A), and high-pH reversed-phase chromatography was employed using an Agilent 1100 HPLC system with an autosampler, a YMC Triart C18 0.5 x 250 mm column, and a fraction collector. The peptides were separated using the following binary buffer system: high-pH Buffer A (20 mM ammonium formate in water, pH 10) and high-pH Buffer B (20 mM ammonium formate pH 10 in 90% acetonitrile) at a flow rate of 12 μL/minute. The gradient duration was a total of 115 min, with mobile phase compositions in %B as follows: 0-5 min at 2%-15%, 5-15 min at 15%-25%, 15-80 min at 25%-45%, 80-85 min at 45%-65%, 85-95 min at 65%-100%, 95-100 min at 100%, 100-101 min at 100%-2%, and 101-115 min at 2%, resulting in 48 fractions after sample concatenation. Samples were acidified with 1% TFA and dried in a speed vac. Peptides reconstituted in 2% acetonitrile, 0.1% formic acid were on-line separated on a C18 50 cm μPAC column using a EASYnLC-1000 system (all Thermo Fisher Scientific) mounted on a Digital PicoView nanospray source (New Objective), connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Peptide elution was achieved using a linear gradient of increasing concentration of acetonitrile in 0.1% formic acid at a flow rate of 500 nl/min: 0-2 min: 2%, 2-8 min: 2-6%, 8-89 min 6-22%, 89-107 min: 22-40%, 107-111 min: 40-80%, 111-115 min: 80% (washing), 115-116 min: 80-2%, 116-120 min: 2% (equilibration). For MS data acquisition in a data-dependent mode, ions for survey MS1 scans were recorded in profile mode in the Orbitrap detector at a resolution of 120,000 in the range of 400-1400 m/z every 3 seconds for a maximum of 100 ms to reach the normalized AGC target of 50%. The most abundant precursors were selected in the quadrupole within an isolation window of 0.7 m/z for MS2 HCD fragmentation based on advanced peak determination, monoisotopic peak determination set to peptides, within charge states 2-8, dynamic exclusion of 60 s within +/-10 ppm tolerance for isotopes and different charge states, with a minimum intensity of 5e4, with normalized collision energy 34% for a maximum injection time of 200 ms to reach the normalized AGC target of 80%. MS2 HCD spectra were and recorded in the “normal” range, starting at 100 m/z at 50,000 resolution in the Orbitrap analyzer in centroid mode.
2025-08-21 | PXD054143 | Pride
Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.
2022-02-24 | PXD012490 | Pride
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:A total of thirty-two samples mouse serum samples were analyzed in duplicate (64 total injections) using an LC-MS system comprised of a nanoAcquity LC instrument (Waters nanoAcquity UPLC system, Milford, MA) connected to an Orbitrap Fusion Lumos tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA). Peptides were trapped on a nanoAcquity trap column (180 um x 20 mm), washed with 99.5% mobile phase A (0.1% formic acid in water) and separated on a nanoAcquity C18 column (1.7 um BEH130Angstrom, 100 um x 100 mm) at 40 C. Peptides were eluted with a linear gradient of 3% to 40% mobile phase B (0.1 % FA in acetonitrile) over 90 min. Gradient changes were followed at 91min to 85% B and then increased to 95% B at 95 min. The gradient was changed back to 3% of B to equilibrate for 20 minutes prior to the next injection. Each injection consisted of 1.5 ug of purified peptides and injection order was randomized to minimize bias.
Eluted peptides were ionized in positive ion polarity at a 2.1 kV of spraying voltage. MS1 full scans were recorded in the range of m/z 380 to 1580 with a resolution of 120,000 at 200 m/z using the Orbitrap mass analyzer. Automatic gain control was set at 8 x 105 with 50 ms of maximum injection time. Top speed (3sec) data dependent acquisition mode was used to maximize the number of MS2 spectra from each cycle. Higher-energy C-trap dissociation (HCD) was used to fragment selected precursor ions with a normalized collision energy of 30%. Tandem MS scans were performed in the ion trap mass analyzer with 35 ms maximum injection time, an ion target value set at 5 x 103, and dynamic exclusion set to 30s.
2019-10-04 | MSV000084416 | MassIVE
Project description:For LC-MS/MS measurements, protein samples (20 µg/lane) were first submitted to SDS-PAGE. The gel was then stained with Bio-SafeTM Coomassie stain (Bio-Rad), and de-stained with water. Then, protein bands corresponding to the molecular weight of the protein of interest were cut from the polyacrylamide gel and the gel fragments were digested using an in-gel tryptic digestion kit (Thermo Scientific: p/n 89871). Following digestion, extracted peptides were then desalted using PierceTM pipette tips (ThermoFisher) and dried, resuspended in 20 µL of 0.5% acetic acid (pH4.5) and then subjected to LC-MS/MS using an Thermo Scientific Orbitrap Eclipse Tribrid Mass Spectrometer (MS; Thermo Scientific) with an Ultimate 3000 nano-liquid chromatography (nano-LC) and a FAIMS Pro Interface (Thermo Scientific). The LC-MS/MS analysis was performed using an Orbitrap Eclipse MS (Thermo Scientific). Peptides were first loaded onto a trap column (PepMap C18) and then separated by an analytical column (PepMap C18, 2.0 um; 15 cm x 75mm I.D.; Thermo Scientific) at 300 nl/min flow rate using a binary buffer system (buffer A, 0.1% formic acid in water; buffer B, 0.1% formic acid in acetonitrile) with a 165-min gradient (1% to 10% in 8 min; then to 25% buffer B over 117 min; 25% to 32% buffer B in 10 min, then to 95% buffer B over 3 min; back to 1% B in 5 min, and stay equilibration at 1% B for 20 min). Multiple CVs (-40, -55 and -75) were applied for FAIMS separation. For all experiments, the survey scans (MS1) were acquired over a mass range of 375-1500 m/z at a resolution of 60,000 in the Orbitrap. The maximum injection time was set to Dynamic, and AGC target was set to Standard. Monoisotopic peak selection was set to Peptides, and the charge state filter was set to 2-7. For MS/MS acquisition, precursors were isolated with a width of 1.6 m/z, fragmented with HCD using 30% collision energy with a maximum injection time of 100 ms, and collected in Orbitrap at 15,000 resolution. The dynamic exclusion was set to 60 s, and can be shared across different FAIMS experiments. LC-MS/MS data was collected in n=3 independent biological replicates. Proteomic analysis was performed in the MaxQuant-Andromeda software suite (version 1.6.3.4) with most of the default parameters51. An E. coli reference proteome (strain BL21-DE3; taxonomy_id:469008) was used for database search. Other parameters include: trypsin as an enzyme with maximally two missed cleavage sites; protein N-terminal acetylation, methionine oxidation, and pA-Phe and pN-Phe substitution for tyrosine were entered as variable modifications; cysteine carbamidomethylation as a fixed modification; and peptide length was set to a minimum of 7 amino acids. The false-discovery rate of high-confidence protein and peptide identification was 1%. Peptide intensity values derived from MaxQuant were used for quantification. To obtain the MS/MS spectra of peptides, additional analysis was performed using Proteome Discoverer software (version 3.0; Thermo Fisher). Raw data was searched against the E. coli reference proteome (strain BL21(DE3)) with NCBI reference Proteome (469008) using Sequest HT Processor and CWF Basic analysis workflows. Iodoacetamide-mediated cysteine carbamidomethylation was set as a static modification, while methionine oxidation and pA-Phe and pN-Phe substitution for tyrosine were entered as dynamic modifications. Precursor mass tolerance was set at 10?ppm while allowing fragment ions to have a mass deviation of 0.02?Da for the HCD data. Validation of peptide-spectrum matches (PSM) based on q value was done using Percolator, with target false-discovery rates (FDR) of 1% and 5% for stringent and relaxed validations, respectively.
2023-02-28 | MSV000091379 | MassIVE