Neuronal CM samples were subjected to SDS-PAGE, followed by in-gel trypsin digestion (Shevchenko et al., 2006 (link)). The extracted peptides were analyzed on an Orbitrap Fusion Lumos Tribrid Mass Spectrometer coupled with the UltiMate 3000 RSLCnano liquid chromatography system (Thermo Fisher Scientific). The peptides were loaded on Acclaim PepMap100 Nano-Trap Column (100 μm × 2 cm, Thermo Fisher Scientific). Peptides were resolved at 300-nl/min flow rate using a linear gradient of 10% to 35% solvent B (0.1% formic acid in 95% acetonitrile) over 95 minutes on an EASY-Spray column (50 cm × 75 μm ID, Thermo Fisher Scientific). MaxQuant (v1.5.5.1) software was used for quantitation and identification of proteins from the mass spectrometry data using mouse UniProt database (released on May 2018) with common contaminant proteins (Cox and Mann, 2008 (link)). Search parameters included, a) trypsin as a proteolytic enzyme with up to 2 missed cleavages; b) first search peptide mass error tolerance of 20 ppm and the main search peptide mass error tolerance of 4 ppm; c) fragment mass error tolerance of 20 ppm; d) carbamidomethylation of cysteine (+57.02146 Da) as a fixed modification: e) oxidation of methionine (+15.99492 Da) and protein acetyl (+42.01056 Da) on N terminus as dynamic modifications. Peptides and proteins were filtered at 1% false-discovery rate.
Acclaim pepmap100 nano trap column
Manufactured by Thermo Fisher Scientific
The Acclaim PepMap100 Nano-Trap Column is a high-performance liquid chromatography (HPLC) column designed for use in nano-scale liquid chromatography applications. The column features a silica-based stationary phase with a particle size of 3 μm and a pore size of 100 Å, providing efficient separation and concentration of peptides and proteins prior to mass spectrometry analysis.
Automatically generated - may contain errors
Lab products found in correlation
Easy spray column, by Thermo Fisher Scientific (4 mentions)
Ultimate 3000 rslcnano liquid chromatography system, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos tribrid mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos tribrid, by Thermo Fisher Scientific (2 mentions)
Ltq velos mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Pepmap rslc, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (1 mentions)
5 protocols using acclaim pepmap100 nano trap column
1
Mass Spectrometry-based Proteomic Workflow
2
Mass Spectrometry-based Proteomic Workflow
3
Shotgun Proteomics by Nano-LC MS/MS
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Samples were analyzed by nano-LC MS/MS by means of a shotgun strategy using a nano-HPLC (EASY-nLC 1000, Thermo Scientific) coupled online to an LTQ Velos mass spectrometer (Thermo Scientific). Tryptic peptides were injected into an Acclaim® PepMap 100 nanotrap column (75 μm x 2 cm, Thermo Scientific) and separated on a 50 μm x 15 cm C18 Easy Spray column (PepMap® RSLC, 2 μM, 100 Å, Thermo Scientific) at a constant flow rate of 250 nL/min at 45°C. Peptide elution was achieved with a 70 min gradient from 0% to 55% mobile phase B (A: 0.1% formic acid; B: 0.1% formic acid in acetonitrile). Online MS analysis was carried out in data-dependent acquisition mode in two steps: acquisition of full MS scans in positive ion mode with an m/z range from 400 to 1200 Da followed by CID fragmentation of the ten most intense ions using a dynamic exclusion list (exclusion duration 45 s). The following parameters were set: spray voltage, 2.3 kV; capillary temperature, 260°C; normalized collision energy, 35; activation Q energy, 0.25; and activation time, 15 ms.
4
Phosphotyrosine Peptide Analysis by Orbitrap MS
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The phosphotyrosine peptides and non-phosphorylated peptides were analyzed on Orbitrap Fusion Lumos Tribrid (Thermo Scientific, San Jose, CA, USA) coupled with Easy-nanoLC 1200 nanoflow liquid chromatography system (Thermo Scientific). The peptides from each fraction were reconstituted in 10% formic acid and loaded on Acclaim PepMap100 Nano Trap Column (100 μm × 2 cm) (Thermo Scientific) packed with 5 μm C18 particles at a flow rate of 5 μl per minute. Peptides were resolved at 250 nl/min flow rate using a linear gradient of 10–35% solvent B (0.1% formic acid in 95% acetonitrile) over 95 min on the EASY-Spray column (50 cm × 75 µm ID, PepMap RSLC C18, and 2 µm C18 particles) (Thermo Scientific) and it was fitted on EASY-Spray ion source that was operated at 2.0 kV voltage. Mass spectrometry analysis was carried out in a data-dependent manner with full scans in the range of m/z 350 to 1500. Both MS and MS/MS were acquired and measured using Orbitrap mass analyzer. Full MS scans were measured at a resolution of 120,000 at m/z 200. Precursor ions were fragmented using a higher-energy collisional dissociation method and detected at a mass resolution of 30,000 at m/z 200.
5
Targeted LC-MS/MS Proteomics Analysis
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LC-MS/MS analyses were conducted as described previously with minor modifications [30 (link),31 (link)]. The peptide samples were analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer interfaced with an Ultimate 3000 RSLCnano nanoflow liquid chromatography system (Thermo Fisher Scientific). The peptides reconstituted in 15 μL of 0.1% FA were loaded on Acclaim PepMap100 Nano-Trap column (100 μm × 2 cm, Thermo Fisher Scientific) packed with 5 μm C18 particles at a flow rate of 5 μL per min. The flow rate employed was 250 nL/min using a linear gradient of 6% to 28% solvent B (0.1% FA in 95% ACN and 5% water) over 55 min on an EASY-Spray column (50 cm × 75 µm, Thermo Fisher Scientific) packed with 2 µm C18 particles (Thermo Fisher Scientific), which was fitted with an EASY-Spray ion source operated at a voltage of 2.0 kV. Mass spectrometry analysis was conducted in the targeted MS2 mode. The MS1 scan range for a full survey scan was acquired from m/z 300 to 1600 with a resolution of 120,000 at an m/z of 200. The mass resolution for MS2 was set to 30,000 at an m/z of 200. Automatic gain control was set to 500,000 and 100,000 ions for MS1 and MS2, respectively. The maximum ion injection time and HCD fragmentation energy for each peptide are listed in Table S1 .
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